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

2019 ◽  
Vol 366 (14) ◽  
Author(s):  
Amee A Sangani ◽  
Alexandra L McCully ◽  
Breah LaSarre ◽  
James B McKinlay
Keyword(s):  
Escherichia Coli ◽  
Rhodopseudomonas Palustris ◽  
H2 Production ◽  
Substantial Contribution ◽  
Individual Species ◽  
Ammonium Excretion ◽  
E Coli ◽  
Carbon And Nitrogen ◽  
Relative Contribution ◽  
Mutualistic Relationship

ABSTRACT 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 its 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. The level of ammonium excretion influenced the time over which E. coliwas exposed to formate, the types of E. colifermentation products available to R. palustris, and the pH of the medium, all of which affected the contribution of each species to H2 production.

scholarly journals FermentativeEscherichia colimakes a substantial contribution to H2production in coculture with phototrophicRhodopseudomonas palustris

2019 ◽  
Author(s):  
Amee A. Sangani ◽  
Alexandra L. McCully ◽  
Breah LaSarre ◽  
James B. McKinlay
Keyword(s):  
Rhodopseudomonas Palustris ◽  
Substantial Contribution ◽  
Individual Species ◽  
Ammonium Excretion ◽  
E Coli ◽  
Carbon And Nitrogen ◽  
Relative Contribution ◽  
Mutualistic Relationship ◽  
Acidic Conditions ◽  
High Level

AbstractIndividual 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, fermentativeEscherichia coliand phototrophicRhodopseudomonas palustrisexchange essential carbon and nitrogen, respectively, to establish a mutualistic relationship. In this relationship, each species produces H2biofuel as a byproduct of their metabolism. However, the extent to which each species contributes to H2production and the factors that influence their relative contributions were previously unknown. By comparing H2yields in cocultures pairingR. palustriswith either wild-typeE. colior a formate hydrogenlyase mutant that is incapable of H2production, we determined the relative contribution of each species to total H2production. Our results indicate thatE. colicontributes between 32% and 86% of the H2produced in coculture depending on the level of ammonium excreted by theR. palustrispartner. AnR. palustrisstrain that stimulated rapidE. coligrowth through a high level of ammonium excretion resulted in earlier accumulation of formate and acidic conditions that allowedE. colito be the major contributor to H2production.

scholarly journals AnEscherichia colinitrogen starvation response is important for mutualistic coexistence withRhodopseudomonas palustris

2018 ◽  
Author(s):  
Alexandra L. McCully ◽  
Megan G. Behringer ◽  
Jennifer R. Gliessman ◽  
Evgeny V. Pilipenko ◽  
Jeffrey L. Mazny ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Starvation ◽  
Physiological State ◽  
Rhodopseudomonas Palustris ◽  
Genetically Engineered ◽  
Individual Species ◽  
Starvation Response ◽  
E Coli ◽  
Protein Levels ◽  
Stable Coexistence

AbstractMicrobial 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 individual’s 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 fermentativeEscherichia coliand phototrophicRhodopseudomonas 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 N2into NH4+, providingE. coliwith 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 withR. palustris, E. coligene-expression changes resembled a nitrogen starvation response under the control of the transcriptional regulator NtrC. By genetically disruptingE. coliNtrC, we determined that a nitrogen starvation response is important for a stable coexistence, especially at lowR. palustrisNH4+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.ImportanceMutualistic 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 ofRhodopseudomonas palustrisandEscherichia coligrowing cooperatively through bidirectional nutrient exchange, we determined that anE. colinitrogen starvation response is important for maintaining a stable coexistence. The lack of anE. colinitrogen 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.

scholarly journals AnEscherichia coliNitrogen Starvation Response Is Important for Mutualistic Coexistence withRhodopseudomonas palustris

2018 ◽  
Vol 84 (14) ◽  
Author(s):  
Alexandra L. McCully ◽  
Megan G. Behringer ◽  
Jennifer R. Gliessman ◽  
Evgeny V. Pilipenko ◽  
Jeffrey L. Mazny ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Starvation ◽  
Physiological State ◽  
Rhodopseudomonas Palustris ◽  
Genetically Engineered ◽  
Individual Species ◽  
Starvation Response ◽  
Content Type ◽  
E Coli ◽  
Stable Coexistence

ABSTRACTMicrobial 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 individual species' 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 fermentativeEscherichia coliand phototrophicRhodopseudomonas palustris. In this coculture,E. colianaerobically ferments sugars into excreted organic acids as a carbon source forR. palustris. In return, a genetically engineeredR. palustrisstrain constitutively converts N2into NH4+, providingE. coliwith essential nitrogen. Using transcriptome sequencing (RNA-seq) and proteomics, we identified transcript and protein levels that differ in each partner when grown in coculture versus monoculture. When in coculture withR. palustris,E. coligene expression changes resembled a nitrogen starvation response under the control of the transcriptional regulator NtrC. By genetically disruptingE. coliNtrC, we determined that a nitrogen starvation response is important for a stable coexistence, especially at lowR. palustrisNH4+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.IMPORTANCEMutualistic 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 ofRhodopseudomonas palustrisandEscherichia coligrowing cooperatively through bidirectional nutrient exchange, we determined that anE. colinitrogen starvation response is important for maintaining a stable coexistence. The lack of anE. colinitrogen 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.

scholarly journals Enhanced nutrient uptake is sufficient to drive emergent cross-feeding between bacteria in a synthetic community

2019 ◽  
Author(s):  
Ryan K Fritts ◽  
Jordan T Bird ◽  
Megan G Behringer ◽  
Anna Lipzen ◽  
Joel Martin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nutrient Uptake ◽  
Rhodopseudomonas Palustris ◽  
Biogeochemical Cycles ◽  
Wild Type ◽  
Fermentation Products ◽  
E Coli ◽  
Constitutive Activation ◽  
Host Health ◽  
Nitrogen Scavenging

ABSTRACTInteractive microbial communities are ubiquitous, influencing biogeochemical cycles and host health. One widespread interaction is nutrient exchange, or cross-feeding, wherein metabolites are transferred between microbes. Some cross-fed metabolites, such as vitamins, amino acids, and ammonium (NH4+), are communally valuable and impose a cost on the producer. The mechanisms that enforce cross-feeding of communally valuable metabolites are not fully understood. Previously we engineered mutualistic cross-feeding between N2-fixing Rhodopseudomonas palustris and fermentative Escherichia coli. Engineered R. palustris excreted essential nitrogen as NH4+ to E. coli while E. coli excreted essential carbon as fermentation products to R. palustris. Here, we enriched for nascent cross-feeding in cocultures with wild-type R. palustris, not known to excrete NH4+. Emergent NH4+ cross-feeding was driven by adaptation of E. coli alone. A missense mutation in E. coli NtrC, a regulator of nitrogen scavenging, resulted in constitutive activation of an NH4+ transporter. This activity likely allowed E. coli to subsist on the small amount of leaked NH4+ and better reciprocate through elevated excretion of organic acids from a larger E. coli population. Our results indicate that enhanced nutrient uptake by recipients, rather than increased excretion by producers, is an underappreciated yet possibly prevalent mechanism by which cross-feeding can emerge.

scholarly journals Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flasks: Part 1

2016 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Escherichia Coli ◽  
Cell Density ◽  
Yeast Extract ◽  
High Cell Density ◽  
Defined Medium ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
E Coli ◽  
Carbon And Nitrogen ◽  
Shake Flasks

Sufficient quantities of cells of consistent characteristics are needed for studying biological processes (at the population level) in many areas of applied microbiology. However, generating the requisite biomass by cell culture is usually the rate-limiting step of a project given the relatively low biomass yield of many commercial culture media in shake flasks. This work reports the formulation of a semi-defined medium that enabled aerobic high cell density cultivation of Escherichia coli DH5α (ATCC 53868) in shake flasks. The formulated medium (FM) comprises: a buffer system (K2HPO4: 12.54 g/L and KH2PO4: 2.31 g/L); vitamins and trace elements (yeast extract: 12.0 g/L); salts (NaCl: 5.0 g/L and MgSO4: 0.24 g/L); and carbon and nitrogen sources (D-Glucose: 6.0 g/L and NH4Cl: 1.5 g/L). Notable characteristics of this medium are: high buffer capacity (89 mM phosphate), 1:1 molar ratio between D-Glucose and NH4Cl, and yeast extract providing trace elements and a secondary source of carbon and nitrogen. Preliminary data revealed that an OD600nm of 9 was attained after 24 hours of cultivation at 37 oC, with glucose and NH4Cl as the main nutrients. At 48 hours, the OD600nm reached a maximum value of 11 with yeast extract providing the necessary nutrients for cell growth and biomass formation. The broth’s pH varied between 5.5 and 7.8 during cultivation. For comparison, the maximum OD600nm of E. coli grown in three commonly used complex media: Nutrient Broth, LB Lennox, and Tryptic Soy Broth (TSB) were 1.4, 3.2 and 9.2, respectively, under identical culture conditions. Finally, FM maintained the viability of a larger population of cells for three days - compared to a population collapse observed in TSB after one day. Collectively, the present findings suggested that the formulated medium might find use as a high cell density aerobic growth medium for E. coli in shake flasks. Part 2 of this work describes improvements in medium performance - specifically, higher cell yield as well as a shorter diauxic lag phase and total culture period – achieved through a small reduction in D-Glucose and NH4Cl concentrations in the medium composition. An abstract preprint of Part 2 is available at https://peerj.com/preprints/117/

scholarly journals Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flasks: Part 1

2017 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Escherichia Coli ◽  
Cell Density ◽  
Yeast Extract ◽  
High Cell Density ◽  
Defined Medium ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
E Coli ◽  
Carbon And Nitrogen ◽  
Shake Flasks

Sufficient quantities of cells of consistent characteristics are needed for studying biologicalprocesses (at the population level ) in many areas of applied microbiology. However, generating the requisite biomass by cell culture is usually the rate-limiting step of a project given the relatively low biomass yield of many commercial culture media in shake flask culture systems. This work reports the formulation of a semi-defined medium that enabled aerobic high cell density cultivation of Escherichia coli DH5α (ATCC 53868) in shake flasks. The formulated medium (FM) comprises: a buffer system (K2HPO4 : 12.54 g/L and KH2 PO4 : 2.31 g/L); vitamins and trace elements (yeast extract: 12.0 g/L); salts (NaCl: 5.0 g/L and MgSO4 : 0.24 g/L); and carbon and nitrogen sources (D-Glucose: 6.0 g/L and NH4Cl: 1.5 g/L). Notable characteristics of this medium are: high buffer capacity (89 mM phosphate), 1:1 molar ratio between D-Glucose and NH4Cl, and yeast extract providing trace elements and a secondary source of carbon and nitrogen. Preliminary data revealed an OD 600nm of 9 after 24 hours of cultivation at 37 oC, presumably with glucose and NH4Cl as the main nutrients. At 48 hours, an OD 600nm of 11 was attained with yeast extract providing the necessary nutrients for cell growth and biomass formation. The broth’s pH varied between 5.5 and 7.8 during cultivation. On the other hand, the maximum OD 600nm of E. coli grown in three commonly used complex media: Nutrient Broth, LB Lennox, and Tryptic Soy Broth (TSB) were 1.4, 3.2 and 9.2, respectively, under identical culture conditions. Finally, FM maintained the viability of a larger population of cells for three days, compared to a population collapse in TSB broth after one day. Collectively, the results suggested that the formulated medium might find use as a high cell density aerobic growth medium for E. coli in shake flasks. Part 2 of this work describes improvements in medium performance ; specifically, higher cell yield as well as a shorter diauxic lag phase and total culture period achieved through a small reduction in D-Glucose and NH4Cl concentrations in the medium composition. An abstract preprint of Part 2 is available at https://peerj.com/preprints/117/

scholarly journals Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flask culture system

2015 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Escherichia Coli ◽  
Cell Density ◽  
Yeast Extract ◽  
High Cell Density ◽  
Growth Media ◽  
Buffer System ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
E Coli ◽  
Carbon And Nitrogen

Microbes in environmental studies should be cultured in growth media with characteristics as close to their original habitat as possible, and which also allows a high cell density to be attained for providing enough cells in subsequent experiments. This in-progress report describes the formulation of a medium with an environmentally-relevant composition, and which also affords aerobic high cell density cultivation of Escherichia coli DH5α in shake flasks. The formulated medium comprises four components: a buffer system (K2HPO4: 12.54 g/L and KH2PO4: 2.31 g/L), vitamins (yeast extract: 12.0 g/L), salts (NaCl: 5.0 g/L and MgSO4: 0.24 g/L), and carbon and nitrogen sources (D-Glucose: 6.0 g/L and NH4Cl: 1.5 g/L). Notable characteristics of this medium were: high capacity phosphate buffer system (89 mM phosphate); 1:1 molar ratio between D-Glucose and NH4Cl; and yeast extract providing trace elements and a secondary carbon and nitrogen source. Growth experiments revealed that an OD600nm of 9 was attained after 24 hours of cultivation at 37 oC. This phase of growth was largely fuelled by glucose and NH4Cl. After 48 hours, the OD600nm reached 11, which was fuelled by the mixture of carbohydrates, lipids and proteins in yeast extract. Broth’s pH varied between 5.5 and 7.8 during cultivation, which was in the range conducive for growth of E. coli. In comparison, the OD600nm of E. coli reached 1.4, 3.2, and 9.2 for three commonly used complex media; Nutrient Broth, LB Lennox, and Tryptic Soy Broth, respectively, over 48 hours under identical culture conditions. In addition, the formulated medium was able to maintain a large viable cell population for a longer period of time (three days) relative to Tryptic Soy Broth. Thus, preliminary data suggested that the formulated medium holds potential for use as a high cell density aerobic growth medium for Gram-negative bacteria.

scholarly journals Construction of Direct Z-Scheme Photocatalyst by Mg1.2Ti1.8O5 and g-C3N4 Nanosheets toward Photocatalytic H2 Production and Disinfection

2019 ◽  
Vol 2019 ◽  
pp. 1-9
Author(s):  
Sijia Gu ◽  
Dan Zhang ◽  
Shirong Luo ◽  
Heng Yang
Keyword(s):  
Escherichia Coli ◽  
Photocatalytic Performance ◽  
Diffuse Reflectance Spectroscopy ◽  
Sol Gel ◽  
H2 Production ◽  
X Ray ◽  
E Coli ◽  
Carrier Separation ◽  
Positive Valence ◽  
Calcination Method

Exploring a novel and efficient photocatalyst is the key research goal to relieve energy and environmental issues. Herein, Z-scheme heterojunction composites were successfully fabricated by loading g-C3N4 nanosheets (CN) on the surface of Mg1.2Ti1.8O5 nanoflakes (MT) through a simple sol-gel method followed by the calcination method. The crystalline phase, morphologies, specific surface area, and optical and electrochemical performance of the samples were characterized by X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), energy-disperse X-ray spectroscopy (EDS), Brunauer-Emmett-Teller (BET), diffuse reflectance spectroscopy (DRS), and electrochemical measurements. Considering the suitable band structures of the components, the photocatalytic performance was evaluated by photocatalytic H2O splitting and photocatalytic inactivation of Escherichia coli (E. coli). Among the samples, MT/CN-10 (the molar percentage of melamine to as-obtained Mg-Ti gel was 10%) shows superior photocatalytic performance, which the average H2 production rate was 3.57 and 7.24 times higher than those of MT and CN alone. Additionally, the efficiency of inactivating Escherichia coli (E. coli) over MT/CN-10 was 1.95 and 2.06 times higher as compared to pure MT and CN, respectively. The enhancement of the photocatalytic performance was attributed to the advantages of the extremely negative conduction band (CB) of CN and the extremely positive valence band (VB) of MT, the enhanced light absorption, and more efficient photogenerated charge carrier separation.

scholarly journals Covert Cross-Feeding Revealed by Genome-Wide Analysis of Fitness Determinants in a Synthetic Bacterial Mutualism

2020 ◽  
Vol 86 (13) ◽  
Author(s):  
Breah LaSarre ◽  
Adam M. Deutschbauer ◽  
Crystal E. Love ◽  
James B. McKinlay
Keyword(s):  
Escherichia Coli ◽  
Microbial Communities ◽  
Rhodopseudomonas Palustris ◽  
Microbial Interactions ◽  
Dependent Manner ◽  
Fermentation Products ◽  
Content Type ◽  
E Coli ◽  
Cellular Processes ◽  
Genome Wide

ABSTRACT Microbial interactions abound in natural ecosystems and shape community structure and function. Substantial attention has been given to cataloging mechanisms by which microbes interact, but there is a limited understanding of the genetic landscapes that promote or hinder microbial interactions. We previously developed a mutualistic coculture pairing Escherichia coli and Rhodopseudomonas palustris, wherein E. coli provides carbon to R. palustris in the form of glucose fermentation products and R. palustris fixes N2 gas and provides nitrogen to E. coli in the form of NH4+. The stable coexistence and reproducible trends exhibited by this coculture make it ideal for interrogating the genetic underpinnings of a cross-feeding mutualism. Here, we used random barcode transposon sequencing (RB-TnSeq) to conduct a genome-wide search for E. coli genes that influence fitness during cooperative growth with R. palustris. RB-TnSeq revealed hundreds of genes that increased or decreased E. coli fitness in a mutualism-dependent manner. Some identified genes were involved in nitrogen sensing and assimilation, as expected given the coculture design. The other identified genes were involved in diverse cellular processes, including energy production and cell wall and membrane biogenesis. In addition, we discovered unexpected purine cross-feeding from R. palustris to E. coli, with coculture rescuing growth of an E. coli purine auxotroph. Our data provide insight into the genes and gene networks that can influence a cross-feeding mutualism and underscore that microbial interactions are not necessarily predictable a priori. IMPORTANCE Microbial communities impact life on Earth in profound ways, including driving global nutrient cycles and influencing human health and disease. These community functions depend on the interactions that resident microbes have with the environment and each other. Thus, identifying genes that influence these interactions will aid the management of natural communities and the use of microbial consortia as biotechnology. Here, we identified genes that influenced Escherichia coli fitness during cooperative growth with a mutualistic partner, Rhodopseudomonas palustris. Although this mutualism centers on the bidirectional exchange of essential carbon and nitrogen, E. coli fitness was positively and negatively affected by genes involved in diverse cellular processes. Furthermore, we discovered an unexpected purine cross-feeding interaction. These results contribute knowledge on the genetic foundation of a microbial cross-feeding interaction and highlight that unanticipated interactions can occur even within engineered microbial communities.

Overview of Verotoxigenic Escherichia coli†

1997 ◽  
Vol 60 (11) ◽  
pp. 1444-1446 ◽  
Author(s):  
MARGUERITE A. NEILL
Keyword(s):  
Escherichia Coli ◽  
Control Strategies ◽  
Animal Origin ◽  
Research Needs ◽  
Apple Cider ◽  
E Coli ◽  
Relative Contribution ◽  
Uremic Syndrome ◽  
Human Illness ◽  
Developed World

Verotoxigenic Escherichia coli are a recently described class of diarrheagenic E. coli. The most prominent member of this group, serotype O157:H7, is a well-recognized cause of hemorrhagic colitis and hemolytic uremic syndrome. This serious human pathogen has caused numerous outbreaks in the developed world and has contaminated such widely disparate foods as ground beef, apple cider, and lettuce. Serotypes other than O157:H7 have also been found to cause sporadic enteric disease and several outbreaks have been recently described. The non-O157 SLTEC are more frequently present in food animals and foods of animal origin than serotype O157:H7. Particular non-O157 serotypes (such as O26) have a definite association with HUS. Surveillance data from several regions suggests that there may be important differences in the distribution of serotypes causing HUS in different geographic areas. While more than 100 serotypes of E. coli have been identified as possessing one or more SLT genes, far fewer than that number have been convincingly associated with human illness. Current research needs to determine those additional virulence traits which confer pathogenicity on organisms possessing the SLT gene. Equally important will be to ascertain the relative contribution of different serotypes to human disease in order to develop sound, scientifically based, control strategies.

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