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 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 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.

scholarly journals Engineering Escherichia coli for Microbial Production of Butanone

2016 ◽  
Vol 82 (9) ◽  
pp. 2574-2584 ◽  
Author(s):  
Kajan Srirangan ◽  
Xuejia Liu ◽  
Lamees Akawi ◽  
Mark Bruder ◽  
Murray Moo-Young ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Batch Cultivation ◽  
Culture Conditions ◽  
Sleeping Beauty ◽  
Genetically Engineered ◽  
Content Type ◽  
Factors Affecting ◽  
Formation Pathway ◽  
E Coli ◽  
Biochemical Genetic

ABSTRACTTo expand the chemical and molecular diversity of biotransformation using whole-cell biocatalysts, we genetically engineered a pathway inEscherichia colifor heterologous production of butanone, an important commodity ketone. First, a 1-propanol-producingE. colihost strain with its sleeping beauty mutase (Sbm) operon being activated was used to increase the pool of propionyl-coenzyme A (propionyl-CoA). Subsequently, molecular heterofusion of propionyl-CoA and acetyl-CoA was conducted to yield 3-ketovaleryl-CoA via a CoA-dependent elongation pathway. Lastly, 3-ketovaleryl-CoA was channeled into the clostridial acetone formation pathway for thioester hydrolysis and subsequent decarboxylation to form butanone. Biochemical, genetic, and metabolic factors affecting relative levels of ketogenesis, acidogenesis, and alcohologenesis under selected fermentative culture conditions were investigated. Using the engineeredE. colistrain for batch cultivation with 30 g liter−1glycerol as the carbon source, we achieved coproduction of 1.3 g liter−1butanone and 2.9 g liter−1acetone. The results suggest that approximately 42% of spent glycerol was utilized for ketone biosynthesis, and thus they demonstrate potential industrial applicability of this microbial platform.

scholarly journals The Adaptive Response to Long-Term Nitrogen Starvation in Escherichia coli Requires the Breakdown of Allantoin

2020 ◽  
Vol 202 (17) ◽  
Author(s):  
Amy Switzer ◽  
Lynn Burchell ◽  
Josh McQuail ◽  
Sivaramesh Wigneshweraraj
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Starvation ◽  
Nutrient Starvation ◽  
Continuous Growth ◽  
Content Type ◽  
E Coli ◽  
Transcriptional Changes ◽  
N Starvation ◽  
Over Time

ABSTRACT Bacteria initially respond to nutrient starvation by eliciting large-scale transcriptional changes. The accompanying changes in gene expression and metabolism allow the bacterial cells to effectively adapt to the nutrient-starved state. How the transcriptome subsequently changes as nutrient starvation ensues is not well understood. We used nitrogen (N) starvation as a model nutrient starvation condition to study the transcriptional changes in Escherichia coli experiencing long-term N starvation. The results reveal that the transcriptome of N-starved E. coli undergoes changes that are required to maximize chances of viability and to effectively recover growth when N starvation conditions become alleviated. We further reveal that, over time, N-starved E. coli cells rely on the degradation of allantoin for optimal growth recovery when N becomes replenished. This study provides insights into the temporally coordinated adaptive responses that occur in E. coli experiencing sustained N starvation. IMPORTANCE Bacteria in their natural environments seldom encounter conditions that support continuous growth. Hence, many bacteria spend the majority of their time in states of little or no growth due to starvation of essential nutrients. To cope with prolonged periods of nutrient starvation, bacteria have evolved several strategies, primarily manifesting themselves through changes in how the information in their genes is accessed. How these coping strategies change over time under nutrient starvation is not well understood, and this knowledge is important not only to broaden our understanding of bacterial cell function but also to potentially find ways to manage harmful bacteria. This study provides insights into how nitrogen-starved Escherichia coli bacteria rely on different genes during long-term nitrogen starvation.

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.

Biodecomposition of Phenanthrene and Pyrene by a Genetically Engineered Escherichia coli

2020 ◽  
Vol 14 (2) ◽  
pp. 121-133 ◽  
Author(s):  
Maryam Ahankoub ◽  
Gashtasb Mardani ◽  
Payam Ghasemi-Dehkordi ◽  
Ameneh Mehri-Ghahfarrokhi ◽  
Abbas Doosti ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Performance ◽  
Human Cancer ◽  
Genetically Engineered ◽  
Hazardous Substances ◽  
E Coli ◽  
Spiked Soil ◽  
Engineered Escherichia Coli ◽  
Genetically Manipulated ◽  
Hplc Measurement

Background: Genetically engineered microorganisms (GEMs) can be used for bioremediation of the biological pollutants into nonhazardous or less-hazardous substances, at lower cost. Polycyclic aromatic hydrocarbons (PAHs) are one of these contaminants that associated with a risk of human cancer development. Genetically engineered E. coli that encoded catechol 2,3- dioxygenase (C230) was created and investigated its ability to biodecomposition of phenanthrene and pyrene in spiked soil using high-performance liquid chromatography (HPLC) measurement. We revised patents documents relating to the use of GEMs for bioremediation. This approach have already been done in others studies although using other genes codifying for same catechol degradation approach. Objective: In this study, we investigated biodecomposition of phenanthrene and pyrene by a genetically engineered Escherichia coli. Methods: Briefly, following the cloning of C230 gene (nahH) into pUC18 vector and transformation into E. coli Top10F, the complementary tests, including catalase, oxidase and PCR were used as on isolated bacteria from spiked soil. Results: The results of HPLC measurement showed that in spiked soil containing engineered E. coli, biodegradation of phenanthrene and pyrene comparing to autoclaved soil that inoculated by wild type of E. coli and normal soil group with natural microbial flora, were statistically significant (p<0.05). Moreover, catalase test was positive while the oxidase tests were negative. Conclusion: These findings indicated that genetically manipulated E. coli can provide an effective clean-up process on PAH compounds and it is useful for bioremediation of environmental pollution with petrochemical products.

scholarly journals Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

2012 ◽  
Vol 78 (15) ◽  
pp. 5238-5246 ◽  
Author(s):  
Dongfei Han ◽  
Ji-Young Ryu ◽  
Robert A. Kanaly ◽  
Hor-Gil Hur
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Hypothetical Protein ◽  
Aldehyde Group ◽  
Agrobacterium Vitis ◽  
T7 Promoter ◽  
Content Type ◽  
E Coli ◽  
Cell Extracts ◽  
Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

scholarly journals Autotransporter Protein-Encoding Genes of Diarrheagenic Escherichia coli Are Found in both Typical and Atypical Enteropathogenic E. coli Strains

2012 ◽  
Vol 79 (1) ◽  
pp. 411-414 ◽  
Author(s):  
Afonso G. Abreu ◽  
Vanessa Bueris ◽  
Tatiane M. Porangaba ◽  
Marcelo P. Sircili ◽  
Fernando Navarro-Garcia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Content Type ◽  
E Coli ◽  
Diarrheagenic Escherichia Coli ◽  
Virulence Markers ◽  
Protein Encoding ◽  
Encoding Genes ◽  
Specific Virulence

ABSTRACTAutotransporter (AT) protein-encoding genes of diarrheagenicEscherichia coli(DEC) pathotypes (cah,eatA,ehaABCDJ,espC,espI,espP,pet,pic,sat, andtibA) were detected in typical and atypical enteropathogenicE. coli(EPEC) in frequencies between 0.8% and 39.3%. Although these ATs have been described in particular DEC pathotypes, their presence in EPEC indicates that they should not be considered specific virulence markers.

scholarly journals A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

2016 ◽  
Vol 60 (10) ◽  
pp. 5995-6002 ◽  
Author(s):  
Kristin R. Baker ◽  
Bimal Jana ◽  
Henrik Franzyk ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Throughput Screening ◽  
Gram Negative Bacteria ◽  
Chromogenic Substrate ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Screening Platform

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

scholarly journals A Rhodobacter sphaeroides Protein Mechanistically Similar to Escherichia coli DksA Regulates Photosynthetic Growth

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Christopher W. Lennon ◽  
Kimberly C. Lemmer ◽  
Jessica L. Irons ◽  
Max I. Sellman ◽  
Timothy J. Donohue ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Structure Function ◽  
Rhodobacter Sphaeroides ◽  
Fatty Acid Content ◽  
Regulatory Protein ◽  
Growth Conditions ◽  
Globular Domain ◽  
Content Type ◽  
E Coli

ABSTRACTDksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the “stringent response” to nutrient starvation in the gammaproteobacteriumEscherichia coliand for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly relatedRhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length toE. coliDksA but lacks the Zn finger motif of theE. coliDksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of anE. colistrain lacking thedksAgene and modulates transcriptionin vitrowithE. coliRNA polymerase (RNAP) similarly toE. coliDksA. RSP2654 reduces RNAP-promoter complex stabilityin vitrowith RNAPs fromE. coliorR. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity toE. coliDksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsphas distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp.IMPORTANCEThe role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis inRhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

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