Engineered Living Structures with Shape-Morphing Capability Enabled by 4D Printing with Functional Bacteria.

Engineered living structures with the incorporation of functional bacteria have been explored extensively in recent years and have shown promising potential applications in biosensing, environmental remediation, and biomedicine. However, it is still rare and challenging to achieve multifunctional capabilities such as material production, shape transformation, and sensing in a single-engineered living structure. In this study, we demonstrate bifunctional living structures by synergistically integrating cellulose-generating bacteria with pH-responsive hydrogels, and the entire structures can be precisely fabricated by three-dimensional (3D) printing. Such 3D-printed bifunctional living structures produce cellulose nanofibers in ambient conditions and have reversible and controlled shape-morphing properties (usually referred to as four-dimensional printing). Those functionalities make them biomimetic versions of silkworms in the sense that both can generate nanofibers and have body motion. We systematically investigate the processing-structure-property relationship of the bifunctional living structures. The on-demand separation of 3D cellulose structures from the hydrogel template and the living nature of the bacteria after processing and shape transformation are also demonstrated.

Designed Microbial Biosynthesis of Hierarchical Bone-Mimetic Biocomposites in 3D-Printed Soft Bioreactors.

The creation of 3D biomimetic composite structures has important applications in tissue engineering, lightweight structures, drug delivery, and sensing. Previous approaches in fabricating 3D biomimetic composites have relied on blending or assembling chemically synthesized molecules or structures, making it challenging to achieve precise control of the size, geometry, and internal structure of the biomimetic composites. Here, we present a new approach for the creation of 3D bone-mimetic biocomposites with precisely controlled shape, hierarchical structure, and functionalities. Our approach is based on the integration of programmable microbial biosynthesis with 3D printing of gas-permeable and customizable bioreactors. The organic and inorganic components are bacterial cellulose and calcium hydroxyapatite via a mineral precursor, which are generated by Komagataeibacter xylinus and Bacillus simplex P6A, respectively, in 3D-printed silicone bioreactors in consecutive culturing cycles. This study is of high significance to biocomposites, biofabrication, and tissue engineering as it paves the way for the synergistic integration of microbial biosynthesis and additive manufacturing.

Microbial products linked to steatohepatitis are reduced by deletion of nuclear hormone receptor SHP in mice.

Deletion of the nuclear hormone receptor small heterodimer partner (Shp) ameliorates the development of obesity and nonalcoholic steatohepatitis (NASH) in mice. Liver-specific SHP plays a significant role in this amelioration. The gut microbiota has been associated with these metabolic disorders, and the interplay between bile acids (BAs) and gut microbiota contributes to various metabolic disorders. Since hepatic SHP is recognized as a critical regulator in BA synthesis, we assessed the involvement of gut microbiota in the antiobesity and anti-NASH phenotype of Shp-/- mice. Shp deletion significantly altered the levels of a few conjugated BAs. Sequencing the 16S rRNA gene in fecal samples collected from separately housed mice revealed apparent dysbiosis in Shp-/- mice. Cohousing Shp-/- mice with WT mice during a Western diet regimen impaired their metabolic improvement and effectively disrupted their distinctive microbiome structure, which became indistinguishable from that of WT mice. While the Western diet challenge significantly increased lipopolysaccharide and phenylacetic acid (PAA) levels in the blood of WT mice, their levels were not increased in Shp-/- mice. PAA was strongly associated with hepatic peroxisome proliferator-activated receptor gamma isoform 2 (Pparg2) activation in mice, which may represent the basis of the molecular mechanism underlying the association of gut bacteria and hepatic steatosis. Shp deletion reshapes the gut microbiota possibly by altering BAs. While lipopolysaccharide and PAA are the major driving forces derived from gut microbiota for NASH development, Shp deletion decreases these signaling molecules via dysbiosis, thereby partially protecting mice from diet-induced metabolic disorders.

Enhanced terrestrial Fe(II) mobilization identified through a novel mechanism of microbially driven cave formation in Fe(III)-rich rocks.

Most cave formation requires mass separation from a host rock in a process that operates outward from permeable pathways to create the cave void. Given the poor solubility of Fe(III) phases, such processes are insufficient to account for the significant iron formation caves (IFCs) seen in Brazilian banded iron formations (BIF) and associated rock. In this study we demonstrate that microbially-mediated reductive Fe(III) dissolution is solubilizing the poorly soluble Fe(III) phases to soluble Fe(II) in the anoxic zone behind cave walls. The resultant Fe(III)-depleted material (termed sub muros) is unable to maintain the structural integrity of the walls and repeated rounds of wall collapse lead to formation of the cave void in an active, measurable process. This mechanism may move significant quantities of Fe(II) into ground water and may help to explain the mechanism of BIF dissolution and REE enrichment in the generation of canga. The role of Fe(III) reducing microorganism and mass separation behind the walls (outward-in, rather than inward-out) is not only a novel mechanism of speleogenesis, but it also may identify a previously overlooked source of continental Fe that may have contributed to Archaean BIF formation.

Hydrologic Alteration and Enhanced Microbial Reductive Dissolution of Fe(III) (hydr)oxides Under Flow Conditions in Fe(III)-Rich Rocks: Contribution to Cave-Forming Processes.

Previous work demonstrated that microbial Fe(III)-reduction contributes to void formation, and potentially cave formation within Fe(III)-rich rocks, such as banded iron formation (BIF), iron ore and canga (a surficial duricrust), based on field observations and static batch cultures. Microbiological Fe(III) reduction is often limited when biogenic Fe(II) passivates further Fe(III) reduction, although subsurface groundwater flow and the export of biogenic Fe(II) could alleviate this passivation process, and thus accelerate cave formation. Given that static batch cultures are unlikely to reflect the dynamics of groundwater flow conditions in situ, we carried out comparative batch and column experiments to extend our understanding of the mass transport of iron and other solutes under flow conditions, and its effect on community structure dynamics and Fe(III)-reduction. A solution with chemistry approximating cave-associated porewater was amended with 5.0 mM lactate as a carbon source and added to columns packed with canga and inoculated with an assemblage of microorganisms associated with the interior of cave walls. Under anaerobic conditions, microbial Fe(III) reduction was enhanced in flow-through column incubations, compared to static batch incubations. During incubation, the microbial community profile in both batch culture and columns shifted from a Proteobacterial dominance to the Firmicutes, including Clostridiaceae, Peptococcaceae, and Veillonellaceae, the latter of which has not previously been shown to reduce Fe(III). The bacterial Fe(III) reduction altered the advective properties of canga-packed columns and enhanced permeability. Our results demonstrate that removing inhibitory Fe(II) via mimicking hydrologic flow of groundwater increases reduction rates and overall Fe-oxide dissolution, which in turn alters the hydrology of the Fe(III)-rich rocks. Our results also suggest that reductive weathering of Fe(III)-rich rocks such as canga, BIF, and iron ores may be more substantial than previously understood.

Peptidomimetic Polyurethanes Inhibit Bacterial Biofilm Formation and Disrupt Surface Established Biofilms.

Over 80% of all chronic bacterial infections in humans are associated with biofilms, which are surface-associated bacterial communities encased within a secreted exopolysaccharide matrix that can provide resistance to environmental and chemical insults. Biofilm formation triggers broad adaptive changes in the bacteria, allowing them to be almost 1000-fold more resistant to conventional antibiotic treatments and host immune responses. The failure of antibiotics to eliminate biofilms leads to persistent chronic infections and can promote the development of antibiotic-resistant strains. Therefore, there is an urgent need to develop agents that effectively prevent biofilm formation and eradicate established biofilms. Herein, we present water-soluble synthetic peptidomimetic polyurethanes that can disrupt surface established biofilms of Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli, all of which show tolerance to the conventional antibiotics polymyxin B and ciprofloxacin. Furthermore, while these polyurethanes show poor antimicrobial activity against planktonic bacteria, they prevent surface attachment and stimulate bacterial surface motility to inhibit biofilm formation of both Gram-positive and Gram-negative bacteria at subinhibitory concentrations, without being toxic to mammalian cells. Our results show that these polyurethanes show promise as a platform for the development of therapeutics that target biofilms and modulate surface interactions of bacteria for the treatment of chronic biofilm-associated infections and as antibiofilm agents.

Synergism between Rifampicin and Cationic Polyurethanes Overcomes Intrinsic Resistance of Escherichia coli.

Antibiotic-resistant Gram-negative bacteria are emergent pathogens, causing millions of infections worldwide. While there are several classes of antibiotics that are effective against Gram-positive bacteria, the outer membrane (OM) of Gram-negative bacteria excludes high-molecular-weight hydrophobic antibiotics, making these species intrinsically resistant to several classes of antibiotics, including polyketides, aminocoumarins, and macrolides. The overuse of antibiotics such as ß-lactams has also promoted the spread of resistance genes throughout Gram-negative bacteria, including the production of extended spectrum ß-lactamases (ESBLs). The combination of innate and acquired resistance makes it extremely challenging to identify antibiotics that are effective against Gram-negative bacteria. In this study, we have demonstrated the synergistic effect of outer membrane-permeable cationic polyurethanes with rifampicin, a polyketide that would otherwise be excluded by the OM, on different strains of E. coli, including a clinically isolated uropathogenic multidrug-resistant (MDR) E. coli. Rifampicin combined with a low-dose treatment of a cationic polyurethane reduced the MIC in E. coli of rifampicin by up to 64-fold. The compositions of cationic polyurethanes were designed to have low hemolysis and low cell cytotoxicity while maintaining high antibacterial activity. Our results demonstrate the potential to rescue the large number of available OM-excluded antibiotics to target normally resistant Gram-negative bacteria via synergistic action with these cationic polyurethanes, acting as a novel antibiotic adjuvant class.

Genomic characterization of bacteria from the ultra-oligotrophic Madison aquifer: insight into the archetypical LuxI/LuxR and identification of novel LuxR solos.

OBJECTIVES: To characterize the bacterial community of Wind Cave's Madison aquifer through whole-genome sequencing, and to better understand the bacterial ecology by identifying genes involved in acyl-homoserine lactone (AHL) based quorum-sensing (QS) systems. RESULTS: Genome-based taxonomic classification revealed the microbial richness present in the pristine Madison aquifer. The strains were found to span eleven genera and fourteen species, of which eight had uncertain taxonomic classifications. The genomes of strains SD129 and SD340 were found to contain the archetypical AHL QS system composed of two genes, luxI and luxR. Surprisingly, the genomes of strains SD115, SD129, SD274 and SD316 were found to contain one to three luxR orphans (solos). Strain SD129, besides possessing an archetypical AHL QS luxI-luxR pair, also contained two luxR solos, while strain SD316 contained three LuxR solos and no luxI-luxR pairs. The ligand-binding domain of two LuxR solos, one each from strains SD129 and SD316, were found to contain novel substitutions not previously reported, thus may represent two LuxR orphans that detection and response to unknown self-produced signal(s), or to signal(s) produced by other organisms.

Women Are Underrepresented and Receive Differential Outcomes at ASM Journals: a Six-Year Retrospective Analysis.

Despite 50% of biology Ph.D. graduates being women, the number of women that advance in academia decreases at each level (e.g., from graduate to postdoctorate to tenure track). Recently, scientific societies and publishers have begun examining internal submissions data to evaluate representation and evaluation of women in their peer review processes; however, representation and attitudes differ by scientific field, and to date, no studies have investigated academic publishing in the field of microbiology. Using manuscripts submitted between January 2012 and August 2018 to the 15 journals published by the American Society for Microbiology (ASM), we describe the representation of women at ASM journals and the outcomes of their manuscripts. Senior women authors at ASM journals were underrepresented compared to global and society estimates of microbiology researchers. Additionally, manuscripts submitted by corresponding authors that were women received more negative outcomes than those submitted by men. These negative outcomes were somewhat mediated by whether or not the corresponding author was based in the United States and by the type of institution for United States-based authors. Nonetheless, the pattern for women corresponding authors to receive more negative outcomes on their submitted manuscripts held. We conclude with suggestions to improve the representation of women and decrease structural penalties against women.IMPORTANCE Barriers in science and academia have prevented women from becoming researchers and experts that are viewed as equivalent to their colleagues who are men. We evaluated the participation and success of women researchers at ASM journals to better understand their success in the field of microbiology. We found that women are underrepresented as expert scientists at ASM journals. This is, in part, due to a combination of both low submissions from senior women authors and more negative outcomes on submitted manuscripts for women compared to men.

Ancestral Absence of Electron Transport Chains in Patescibacteria and DPANN.

Recent discoveries suggest that the candidate superphyla Patescibacteria and DPANN constitute a large fraction of the phylogenetic diversity of Bacteria and Archaea. Their small genomes and limited coding potential have been hypothesized to be ancestral adaptations to obligate symbiotic lifestyles. To test this hypothesis, we performed cell-cell association, genomic, and phylogenetic analyses on 4,829 individual cells of Bacteria and Archaea from 46 globally distributed surface and subsurface field samples. This confirmed the ubiquity and abundance of Patescibacteria and DPANN in subsurface environments, the small size of their genomes and cells, and the divergence of their gene content from other Bacteria and Archaea. Our analyses suggest that most Patescibacteria and DPANN in the studied subsurface environments do not form specific physical associations with other microorganisms. These data also suggest that their unusual genomic features and prevalent auxotrophies may be a result of ancestral, minimal cellular energy transduction mechanisms that lack respiration, thus relying solely on fermentation for energy conservation.

Spectroscopic Identification of Peptide Chemistry in the Caulobacter crescentus Holdfast.

The bacterium Caulobacter crescentus is known to attach irreversibly to underwater surfaces by utilizing an adhesive structure called the holdfast, which exhibits the greatest known adhesive strength of any organism. The very small size of the holdfast (∼400 nm wide and ∼40 nm high) has made direct chemical analysis difficult, and its structure remains poorly understood. In this study, we employ spectroscopic techniques, including attenuated total reflection infrared spectroscopy (ATR-IR) and X-ray photoelectron spectroscopy, to probe holdfast chemistry. The data indicate the presence of a peptide signal within the holdfast polymer. By comparing the ATR-IR spectrum of the holdfast to peptidoglycan spectra from other bacterial species, we demonstrate the similarity of the holdfast chemistry to that of peptidoglycan, suggesting peptide cross-linking may play a role in holdfast architecture. To probe the molecular groups at the interface, surface-sensitive sum frequency generation spectroscopy was used to show that aromatic and hydroxyl groups related to this protein content at the adhesive interface could be playing a crucial role in adhesion. On the basis of these results, we propose a model of the holdfast architecture with similarities to the peptide cross-linking observed in the peptidoglycan polymer of the bacterial cell wall. These results not only provide information about the development of adhesives that could be based on holdfast chemical architecture but also reveal a potentially yet unexplored biosynthetic pathway in holdfast synthesis that has not yet been revealed by genetic approaches, thereby opening up a potentially new avenue of research in holdfast synthesis.

Antibiotic eluting poly(ester urea) films for control of a model cardiac implantable electronic device infection.

Cardiac implantable electronic device (CIED) infections acquired during or after surgical procedures are a major complication that are challenging to treat therapeutically, resulting in chronic and sometimes fatal infections. Localized delivery of antibiotics at the surgical site could be used to supplement traditional systemic administration as a preventative measure. Herein, we investigate a cefazolin-eluting l-valine poly(ester urea) (PEU) films as a model system for localized antibiotic delivery for CIEDs. Poly(1-VAL-8) PEU was used to fabricate a series of antibiotic-loaded films with varied loading concentrations (2%, 5%, 10% wt/wt) and thicknesses (40 µm, 80 µm, 140 µm). In vitro release measurements show thickness and loading concentration influence the amount and rate of cefazolin release. Group 10%-140 µm (load-thickness) showed 22.5% release of active pharmaceutical ingredient (API) in the first 24 h and 81.2% of cumulative percent release through day 14 and was found most effective in bacterial clearance in vitro. This group was also effective in clearing a bacterial infection in a model in vivo rat study while eliciting a limited inflammatory response. Our results suggest the feasibility of cefazolin-loaded PEU films as an effective sustained release matrix for localized delivery of antibiotics. SIGNIFICANCE STATEMENT: Implant-associated infections acquired during surgical procedures are a major complication that have proven a challenge to treat clinically, resulting in chronic and sometimes fatal infections. In this manuscript, we investigate an antibiotic-eluting L-valine poly(ester urea) (PEU) films as a model system for localized delivery of cefazolin. Significantly, we demonstrate a wide variation in temporal delivery and dosing within this family of PEUs and show that the delivery can be extended by varying the film thickness. The in vivo results show efficacy in an infected wound model and suggest antibiotic loaded PEU films function as an effective sustained release matrix for localized delivery of antibiotics across a number of clinical indications.

Insight into the resistome and quorum sensing system of a divergent Acinetobacter pittii isolate from an untouched site of the Lechuguilla Cave.

Acinetobacter are Gram-negative bacteria belonging to the sub-phyla Gammaproteobacteria, commonly associated with soils, animal feeds and water. Some members of the Acinetobacter have been implicated in hospital-acquired infections, with broad-spectrum antibiotic resistance. Here we report the whole-genome sequence of LC510, an Acinetobacter species isolated from deep within a pristine location of the Lechuguilla Cave. Pairwise nucleotide comparison to three type strains within the genus Acinetobacter assigned LC510 as an Acinetobacter pittii isolate. Scanning of the LC510 genome identified two genes coding for b-lactamase resistance, despite the fact that LC510 was isolated from a portion of the cave not previously visited by humans and protected from anthropogenic input. The ability to produce acyl-homoserine lactone (AHL) signal in culture medium, an observation that is consistent with the identification of the luxI and luxR homologues in its genome, suggests that cell-to-cell communication remains important in an isolated cave ecosystem.

Life in the dark: far-red absorbing cyanobacteria extend photic zones deep into terrestrial caves.

Chlorophyll (Chl) f and d are the most recently discovered chlorophylls, enabling cyanobacteria to harvest near-infrared radiation (NIR) at 700-780 nm for oxygenic photosynthesis. Little is known about the occurrence of these pigments in terrestrial habitats. Here, we provide first details on spectral photon irradiance within the photic zones of four terrestrial cave systems in concert with a detailed investigation of photopigmentation, light reflectance and microbial community composition. We frequently found Chl f and d along the photic zones of caves characterized by low light enriched in NIR and inhabited by cyanobacteria producing NIR-absorbing pigments. Surprisingly, deeper parts of caves still contained NIR, an effect likely attributable to the reflectance of specific wavelengths by the surface materials of cave walls. We argue that the stratification of microbial communities across the photic zones of cave entrances resembles the light-driven species distributions in forests and aquatic environments.

Bacterial Membrane Selective Antimicrobial Peptide-Mimetic Polyurethanes: Structure-Property Correlations and Mechanisms of Action.

The rise in prevalence of antibiotic resistant strains of bacteria is a very significant challenge for treating life-threatening infections worldwide. A source of novel therapeutics that has shown great promise is a class of biomolecules known as antimicrobial peptides. Previously, within our laboratories, we developed a new family of water-soluble antimicrobial polyurethanes that mimic antimicrobial peptides. Within this current investigation, studies were carried out to gain a greater understanding of the structure/property relationships of the polyurethanes. This was achieved by synthesizing a variety of pendant group functionalized polyurethanes and testing their effectiveness as an antimicrobial by carrying out minimum inhibitory concentration testing and determining their compatibility with blood cells. Additionally, insight into the mode of action of the polyurethanes was obtained through experiments using dye encapsulated phospholipids and assays of bacterial cells that indicated the ability of the polyurethanes to penetrate and disrupt membranes. Collectively, the results indicate that the addition of hydrophobic, uncharged polar, and anionic moieties do not have a strong influence on the antimicrobial activity; yet, the addition of hydrophobic groups enhances cytoplasmic membrane disruption, a larger proportion of cationic pendant groups promotes greater outer membrane disruption of Gram negative bacteria, and uncharged polar groups and anionic groups improve compatibility of the polyurethanes with mammalian cells.

Draft Genome Sequences of Five Proteobacteria Isolated from Lechuguilla Cave, New Mexico, USA, and Insights into Taxonomy and Quorum Sensing.

Genomic resources remain scarce for bacteria isolated from oligotrophic caves. We sequenced the genomes of five Proteobacteria isolated from Lechuguilla Cave in New Mexico. Genome-based phylogeny indicates that each strain belongs to a distinct genus. Two Rhizobiaceae isolates possess genomic potential for the biosynthesis of acyl-homoserine lactone.

Structure-Activity Study of Antibacterial Poly(ester urethane)s with Uniform Distribution of Hydrophobic and Cationic Groups.

Infections associated with antibiotic-resistant bacteria have become a threat to the global public health. Antimicrobial polymers, which are synthetic mimics of antimicrobial peptides, have gained increasing attention, as they may have a lower chance of inducing resistance. The cationic-hydrophobic balance and distribution of cationic and hydrophobic moieties of these polymers is known to have a major effect on antimicrobial activity. We studied the properties of a series of facially amphiphilic antimicrobial surfactant-like poly(ester urethane)s with different hydrophobic pendant groups (P1, P2, and P3) and cationic groups distributed uniformly along the polymer chain. These polymers exhibited bactericidal activity against Gram-negative Escherichia coli and Pseudomonas aeruginosa, as well as Gram-positive Staphylococcus aureus and Staphylococcus epidermidis. Microscopy and dye release assays demonstrated that these polymers cause membrane disruption, which is dependent on the cationic-hydrophobic ratio in the polymer. Membrane permeability assays revealed that these polymers can permeabilize the outer membrane of E. coli and damage the cytoplasmic membrane of both E. coli and S. aureus. In addition, our results indicate that the three polymers exhibit a different extent of membrane disruption against E. coli. P1 caused minor damage to the cytoplasmic membrane integrity, but it was able to dissipate the cytoplasmic membrane potential, leading to cell death. P2 and P3 depolarized the cytoplasmic membrane and also caused significant damage to the cytoplasmic membrane. Overall, we showed a new class of broad-spectrum bactericidal polymers whose membrane disrupting ability against E. coli correlates with the structural differences of the hydrophobic pendant groups.

Polyionic Complexed Antibacterial Heparin-Chitosan Particles for Antibiotic Delivery.

Efficient delivery of antibacterial agents directly to sites of tissue injury faces challenges such as poor drug stability and fast degradation by biological mechanisms. Biocompatible nanocarrier systems can help sustain and control the delivery of antibacterial compounds while reducing the chances of antibacterial resistance or accumulation in unwanted tissues. In this study, we report the application of tailored polyionic particles via ionic interactions between negatively charged heparin and positively charge chitosan for efficient encapsulation of polyhexamethylene biguanide (PHMB) antibiotic. Negative zeta potential was required to encapsulate the positively charged PHMB. We demonstrate that the ratio of heparin to chitosan can be employed to create a tuned surface charge and maximize the bonding of the drug of choice as well as appropriate particle distribution and uniform morphology. Different formulations were evaluated in terms of size, polydispersity, surface charge, and morphology. Out of all these formulations, the best, negatively charged formulation at four-parts heparin to one-part chitosan was successfully encapsulated with PHMB and showed a sustained and controlled release in vitro for around 10 days. Reduced toxic responses (around 48% reduction) were observed from PHMB-loaded particles in contact with human dermal fibroblasts as compared to the soluble form of PHMB. Finally, in terms of antibacterial properties, the particles resulted in growth inhibition as well as the direct killing of both Gram-positive (Enterococcus faecalis) and Gram-negative (Escherichia coli) bacterial strains. The minimum inhibitory concentrations required to inhibit bacterial growth were determined by microplate dilution and LIVE/DEAD bacterial evaluation.

High Microbial Diversity Despite Extremely Low Biomass in a Deep Karst Aquifer.

Despite the importance of karst aquifers as a source of drinking water, little is known about the role of microorganisms in maintaining the quality of this water. One of the limitations in exploring the microbiology of these environments is access, which is usually limited to wells and surface springs. In this study, we compared the microbiology of the Madison karst aquifer sampled via the potentiometric lakes of Wind Cave with surface sampling wells and a spring. Our data indicated that only the Streeter Well (STR), which is drilled into the same hydrogeologic domain as the Wind Cave Lakes (WCL), allowed access to water with the same low biomass (1.56-9.25 × 103 cells mL-1). Filtration of ∼300 L of water from both of these sites through a 0.2 µm filter allowed the collection of sufficient cells for DNA extraction, PCR amplification of 16S rRNA gene sequences, and identification through pyrosequencing. The results indicated that bacteria (with limited archaea and no detectable eukaryotic organisms) dominated both water samples; however, there were significant taxonomic differences in the bacterial populations of the samples. The STR sample was dominated by a single phylotype within the Gammaproteobacteria (Order Acidithiobacillales), which dramatically reduced the overall diversity and species richness of the population. In WCL, despite less organic carbon, the bacterial population was significantly more diverse, including significant contributions from the Gammaproteobacteria, Firmicutes, Chloroflexi, Actinobacteria, Planctomycetes, Fusobacter, and Omnitrophica phyla. Comparisons with similar oligotrophic environments suggest that karst aquifers have a greater species richness than comparable surface environs. These data also demonstrate that Wind Cave provides a unique opportunity to sample a deep, subterranean aquifer directly, and that the microbiology of such aquifers may be more complex than previously anticipated.