Biology subjects

McCully, A. L.

Publications and source records attributed to McCully, A. L..

7 recordsLinked to original sources

bioRxiv2022

Double emulsions as a high-throughput enrichment and isolation platform for slower-growing microbes

Our understanding of in situ microbial physiology is primarily based on physiological characterization of fast-growing and readily-isolatable microbes. Microbial enrichments to obtain novel isolates with slower growth rates or physiologies adapted to low nutrient environments are plagued by intrinsic biases for fastest-growing species when using standard laboratory isolation protocols. New cultivation tools to minimize these biases and enrich for less well-studied taxa are needed. In this study, we developed a high-throughput bacterial enrichment platform based on single cell encapsulation and growth within double emulsions (GrowMiDE). We showed that GrowMiDE can cultivate many different microorganisms and enrich for novel taxa that are never observed in traditional batch enrichments. For example, preventing dominance of the enrichment by fast-growing microbes due to nutrient privatization within the double emulsion droplets allowed cultivation of novel Negativicutes and Methanobacteria from stool samples in rich media enrichment cultures. In competition experiments between growth rate and growth yield specialist strains, GrowMiDE enrichments prevented competition for shared nutrient pools and enriched for slower-growing but more efficient strains. Finally, we demonstrated the compatibility of GrowMiDE with commercial fluorescence-activated cell sorting (FACS) to obtain isolates from GrowMiDE enrichments. Together, GrowMiDE + DE-FACS is a promising new high-throughput enrichment platform that can be easily applied to diverse microbial enrichments or screens.

microbiology↗

bioRxiv2022

Systematic Characterization of Double Emulsion Droplets for Biological Applications

Double emulsion droplets (DEs) are water/oil/water droplets that can be sorted via Fluorescence-Activated Cell Sorting (FACS), allowing for new opportunities in high-throughput cellular analysis, enzymatic screening, and synthetic biology. These applications require stable, uniform droplets with predictable microreactor volumes. However, predicting DE droplet size, shell thickness, and stability as a function of flow rate has remained challenging for monodisperse single core droplets and those containing biologically-relevant buffers, which influence bulk and interfacial properties. As a result, developing novel DE-based bioassays has typically required extensive initial optimization of flow rates to find conditions that produce stable droplets of the desired size and shell thickness. To address this challenge, we conducted systematic size parameterization quantifying how differences in flow rates and buffer properties (viscosity and interfacial tension at water/oil interfaces) alter droplet size and stability, across 6 inner aqueous buffers used across applications such as cellular lysis, microbial growth, and drug delivery, quantifying the size and shell thickness of >22,000 droplets overall. We restricted our study to stable single core droplets generated in a 2-step dripping-dripping formation regime in a straightforward PDMS device. Using data from 138 unique conditions (flow rates and buffer composition), we also demonstrated that a recent physically-derived size law of Wang et al1 can accurately predict double emulsion shell thickness for >95% of observations. Finally, we validated the utility of this size law by using it to accurately predict droplet sizes for a novel bioassay that requires encapsulating growth media for bacteria in droplets. This work has the potential to enable new screening-based biological applications by simplifying novel DE bioassay development.

bioengineering↗

bioRxiv2019

The reverse TCA cycle and reductive amino acid synthesis pathways contribute to electron balance in a Rhodospirillum rubrum Calvin cycle mutant

Purple nonsulfur bacteria (PNSB) use light for energy and organic substrates for carbon and electrons when growing photoheterotrophically. This lifestyle generates more reduced electron carriers than are required for biosynthesis, even during consumption of some of the most oxidized organic substrates like malate and fumarate. Excess reduced electron carriers must be oxidized for photoheterotrophic growth to occur. Diverse PNSB commonly rely on the CO2-fixing Calvin cycle to oxidize excess reduced electron carriers. Some PNSB also produce H2 or reduce terminal electron acceptors as alternatives to the Calvin cycle. Rhodospirillum rubrum Calvin cycle mutants defy this trend by growing phototrophically on malate or fumarate without H2 production or access to terminal electron acceptors. We used 13C-tracer experiments to examine how a Rs. rubrum Calvin cycle mutant maintains electron balance under such conditions. We detected the reversal of some TCA cycle enzymes, which carried reductive flux from malate or fumarate to -ketoglutarate. This pathway and the reductive synthesis of amino acids derived from -ketoglutarate are likely important for electron balance, as supplementing the growth medium with -ketoglutarate-derived amino acids prevented Rs. rubrum Calvin cycle mutant growth unless a terminal electron acceptor was provided. Flux estimates also suggested that the Calvin cycle mutant preferentially synthesized isoleucine using the reductive threonine-dependent pathway instead of the less-reductive citramalate-dependent pathway. Collectively, our results suggest that alternative biosynthetic pathways can contribute to electron balance within the constraints of a relatively constant biomass composition.

microbiology↗

bioRxiv2019

Fermentative Escherichia coli makes a substantial contribution to H2 production in coculture with phototrophic Rhodopseudomonas palustris

Individual species within microbial communities can combine their attributes to produce services that benefit society, such as the transformation of renewable resources into valuable chemicals. Under defined genetic and environmental conditions, fermentative Escherichia coli and phototrophic Rhodopseudomonas palustris exchange essential carbon and nitrogen, respectively, to establish a mutualistic relationship. In this relationship, each species produces H2 biofuel as a byproduct of their metabolism. However, the extent to which each species contributes to H2 production and the factors that influence their relative contributions were previously unknown. By comparing H2 yields in cocultures pairing R. palustris with either wild-type E. coli or a formate hydrogenlyase mutant that is incapable of H2 production, we determined the relative contribution of each species to total H2 production. Our results indicate that E. coli contributes between 32% and 86% of the H2 produced in coculture depending on the level of ammonium excreted by the R. palustris partner. An R. palustris strain that stimulated rapid E. coli growth through a high level of ammonium excretion resulted in earlier accumulation of formate and acidic conditions that allowed E. coli to be the major contributor to H2 production.

microbiology↗

bioRxiv2018

An Escherichia coli nitrogen starvation response is important for mutualistic coexistence with Rhodopseudomonas palustris

Microbial mutualistic cross-feeding interactions are ubiquitous and can drive important community functions. Engaging in cross-feeding undoubtedly affects the physiology and metabolism of individual species involved. However, the nature in which an individuals physiology is influenced by cross-feeding and the importance of those physiological changes for the mutualism have received little attention. We previously developed a genetically tractable coculture to study bacterial mutualisms. The coculture consists of fermentative Escherichia coli and phototrophic Rhodopseudomonas palustris. In this coculture, E. coli anaerobically ferments sugars into excreted organic acids as a carbon source for R. palustris. In return, a genetically-engineered R. palustris constitutively converts N2 into NH4+, providing E. coli with essential nitrogen. Using RNA-seq and proteomics, we identified transcript and protein levels that differ in each partner when grown in coculture versus monoculture. When in coculture with R. palustris, E. coli gene-expression changes resembled a nitrogen starvation response under the control of the transcriptional regulator NtrC. By genetically disrupting E. coli NtrC, we determined that a nitrogen starvation response is important for a stable coexistence, especially at low R. palustris NH4+ excretion levels. Destabilization of the nitrogen starvation regulatory network resulted in variable growth trends and in some cases, extinction. Our results highlight that alternative physiological states can be important for survival within cooperative cross-feeding relationships.\n\nImportanceMutualistic cross-feeding between microbes within multispecies communities is widespread. Studying how mutualistic interactions influence the physiology of each species involved is important for understanding how mutualisms function and persist in both natural and applied settings. Using a bacterial mutualism consisting of Rhodopseudomonas palustris and Escherichia coli growing cooperatively through bidirectional nutrient exchange, we determined that an E. coli nitrogen starvation response is important for maintaining a stable coexistence. The lack of an E. coli nitrogen starvation response ultimately destabilized the mutualism and, in some cases, led to community collapse after serial transfers. Our findings thus inform on the potential necessity of an alternative physiological state for mutualistic coexistence with another species compared to the physiology of species grown in isolation.

microbiology↗

bioRxiv2017

Recipient-Biased Competition For A Cross-Fed Nutrient Is Required For Coexistence Of Microbial Mutualists

Many mutualistic microbial relationships are based on nutrient cross-feeding. Traditionally, cross-feeding is viewed as being unidirectional from the producer to the recipient. This is likely true when a producers metabolic waste, such as fermentation products, provides carbon for a recipient. However, in some cases the cross-fed nutrient holds value for both the producer and the recipient. In such cases, there is potential for nutrient reacquisition by producer cells in a population, leading to competition against recipients. Here we investigate the consequences of inter-partner competition for cross-fed nutrients on mutualism dynamics using an anaerobic coculture pairing fermentative Escherichia coli and phototrophic Rhodopseudomonas palustris. In this coculture, E. coli excretes waste organic acids that provide carbon for R. palustris. In return, R. palustris cross-feeds E. coli ammonium (NH4+), a valuable nitrogen compound that both species prefer. To explore the potential for inter-partner competition, we first used a kinetic model to simulate cocultures with varied affinities for NH4+ in each species. The model predicted that inter-partner competition for cross-fed NH4+ could profoundly impact population dynamics. We then experimentally tested the predictions by culturing mutants lacking NH4+ transporters in both NH4+ competition assays and cooperative cocultures. Both theoretical and experimental results indicated that the recipient must have a competitive advantage in acquiring valuable cross-fed NH4+ to avoid collapse of the mutualism. Thus, the very metabolites that form the basis for cooperative cross-feeding can also be subject to competition between mutualistic partners.\n\nSignificanceMutualistic relationships, particularly those based on nutrient cross-feeding, promote stability of diverse ecosystems and drive global biogeochemical cycles. Cross-fed nutrients within these systems can be either waste products valued only by one partner or nutrients that both partners value. Here, we explore how inter-partner competition for a communally-valuable cross-fed nutrient impacts mutualism dynamics. We discovered that mutualism stability necessitates that the recipient have a competitive advantage against the producer in obtaining the cross-fed nutrient. We propose that the requirement for recipient-biased competition is a general rule for mutualistic coexistence based on the transfer of communally valuable resources, microbial or otherwise.

microbiology↗

bioRxiv2016

Growth-independent cross-feeding modifies boundaries for coexistence in a bacterial mutualism

Nutrient cross-feeding can stabilize microbial mutualisms, including those important for carbon cycling in nutrient-limited anaerobic environments. It remains poorly understood how nutrient limitation within natural environments impacts mutualist growth, cross-feeding levels, and ultimately mutualism dynamics. We examined the effects of nutrient limitation within a mutualism using theoretical and experimental approaches with a synthetic anaerobic coculture pairing fermentative Escherichia coli and phototrophic Rhodopseudomonas palustris. In this coculture, E. coli and R. palustris resemble an anaerobic food web by cross-feeding essential carbon (organic acids) and nitrogen (ammonium), respectively. Organic acid cross-feeding stemming from E. coli fermentation can continue in a growth-independent manner during nutrient limitation, while ammonium cross-feeding by R. palustris is growth-dependent. When ammonium cross-feeding was limited, coculture trends changed yet coexistence persisted under both homogenous and heterogenous conditions. Theoretical modeling indicated that growth-independent fermentation was crucial to sustain cooperative growth under conditions of low nutrient exchange. We also show that growth-independent fermentation sets the upper E. coli cell density at which this mutualism is supported. Thus, growth-independent fermentation can conditionally stabilize or destabilize a mutualism, indicating the potential importance of growth-independent metabolism for nutrient-limited mutualistic communities.\n\nConflict of interestThe authors declare no conflict of interest.

microbiology↗