scholarly journals Quantification of macromolecular biomass composition for constraint-based metabolic modeling

2021 ◽  
Author(s):  
Vetle Simensen ◽  
Christian Schulz ◽  
Emil Karlsen ◽  
Signe Bråtelund ◽  
Idun Burgos ◽  
...  
Keyword(s):  
Critical Role ◽  
Biomass Composition ◽  
E Coli ◽  
Metabolic Phenotypes ◽  
Specific Biomass ◽  
Biomass Determination ◽  
Set Up ◽  
K 12 ◽  
Genome Scale ◽  
Biomass Quantification

Genome-scale metabolic models (GEMs) are mathematical representations of metabolism that allow for in silico simulation of metabolic phenotypes and capabilities. A prerequisite for these predictions is an accurate representation of the biomolecular composition of the cell necessary for replication and growth, implemented in GEMs as the so-called biomass objective function (BOF). The BOF contains the metabolic precursors required for synthesis of the cellular macro- and micromolecular constituents (e.g. protein, RNA, DNA), and its composition is highly dependent on the particular organism, strain, and growth condition. Despite its critical role, the BOF is rarely constructed using specific measurements of the modeled organism, drawing the validity of this approach into question. Thus, there is a need to establish robust and reliable protocols for experimental condition-specific biomass determination. Here, we address this challenge by presenting a general pipeline for biomass quantification, evaluating its performance on Escherichia coli K-12 MG1655 sampled during balanced exponential growth under controlled conditions in a batch-fermentor set-up. We significantly improve both the coverage and molecular resolution compared to previously published workflows, quantifying 91.6% of the biomass. Our measurements display great correspondence with previously reported measurements, and we were also able to detect subtle characteristics specific to the particular E. coli strain. Using the modified E. coli GEM iML1515a, we compare the feasible flux ranges of our experimentally determined BOF with the original BOF, finding that the changes in BOF coefficients considerably affect the attainable fluxes at the genome-scale.

scholarly journals Computation of condition-dependent proteome allocation reveals variability in the macro and micro nutrient requirements for growth

2020 ◽  
Author(s):  
Colton J. Lloyd ◽  
Jonathan Monk ◽  
Laurence Yang ◽  
Ali Ebrahim ◽  
Bernhard O. Palsson
Keyword(s):  
Amino Acids ◽  
Growth Conditions ◽  
Anaerobic Growth ◽  
Metabolic Phenotype ◽  
E Coli ◽  
Scale Models ◽  
Protein Components ◽  
K 12 ◽  
Genome Scale ◽  
Prosthetic Groups

AbstractSustaining a robust metabolic network requires a balanced and fully functioning proteome. In addition to amino acids, many enzymes require cofactors (coenzymes and engrafted prosthetic groups) to function properly. Extensively validated genome-scale models of metabolism and gene expression (ME-models) have the unique ability to compute an optimal proteome composition underlying a metabolic phenotype, including the provision of all required cofactors. Here we use the ME-model for Escherichia coli K-12 MG1655 to computationally examine how environmental conditions change the proteome and its accompanying cofactor usage. We found that: (1) The cofactor requirements computed by the ME model mostly agree with the standard biomass objective function used in models of metabolism alone (M models); (2) ME-model computations reveal non-intuitive variability in cofactor use under different growth conditions; (3) An analysis of ME-model predicted protein use in aerobic and anaerobic conditions suggests an enrichment in the use of prebiotic amino acids in the proteins used to sustain anaerobic growth (4) The ME-model could describe how limitation in key protein components affect the metabolic state of E. coli. Genome-scale models have thus reached a level of sophistication where they reveal intricate properties of functional proteomes and how they support different E. coli lifestyles.

scholarly journals Quantifying cumulative phenotypic and genomic evidence for procedural generation of metabolic network reconstructions

2021 ◽  
Author(s):  
Thomas James Moutinho ◽  
Benjamin C Neubert ◽  
Matthew L Jenior ◽  
Jason A. Papin
Keyword(s):  
Metabolic Network ◽  
Metabolic Networks ◽  
Biological Data ◽  
Supporting Evidence ◽  
Phenotypic Data ◽  
E Coli ◽  
Simultaneous Integration ◽  
Improve Model ◽  
K 12 ◽  
Genome Scale

Genome-scale metabolic network reconstructions (GENREs) are valuable tools for understanding microbial community metabolism. The process of automatically generating GENREs includes identifying metabolic reactions supported by sufficient genomic evidence to generate a draft metabolic network. The draft GENRE is then gapfilled with additional reactions in order to recapitulate specific growth phenotypes as indicated with associated experimental data. Previous methods have implemented absolute mapping thresholds for the reactions automatically included in draft GENREs; however, there is growing evidence that integrating annotation evidence in a continuous form can improve model accuracy. There is a need for flexibility in the structure of GENREs to better account for uncertainty in biological data, unknown regulatory mechanisms, and context specificity associated with data inputs. To address this issue, we present a novel method that provides a framework for quantifying combined genomic, biochemical, and phenotypic evidence for each biochemical reaction during automated GENRE construction. Our method, Constraint-based Analysis Yielding reaction Usage across metabolic Networks (CANYUNs), generates accurate GENREs with a quantitative metric for the cumulative evidence for each reaction included in the network. The structure of a CANYUN GENRE allows for the simultaneous integration of three data inputs while maintaining all supporting evidence for biochemical reactions that may be active in an organism. CANYUNs is designed to maximize the utility of experimental and annotation datasets and to ultimately assist in the curation of the reference datasets used for the automatic reconstruction of metabolic networks. We validated CANYUNs by generating an E. coli K-12 model and compared it to the manually curated reconstruction iML1515. Finally, we demonstrated the use of CANYUNs to build a model by generating an E. coli Nissle CANYUN GENRE using novel phenotypic data that we collected. This method may address key challenges for the procedural construction of metabolic networks by leveraging uncertainty and redundancy in biological data.

scholarly journals Assembly of CS1 Pili: the Role of Specific Residues of the Major Pilin, CooA

2006 ◽  
Vol 188 (1) ◽  
pp. 231-239 ◽  
Author(s):  
Angela M. Starks ◽  
Barbara J. Froehlich ◽  
Tamara N. Jones ◽  
June R. Scott
Keyword(s):  
Diarrheal Disease ◽  
Sequence Similarity ◽  
Critical Role ◽  
Human Pathogens ◽  
Conserved Residues ◽  
Alanine Substitution ◽  
E Coli ◽  
Assembly Pathway ◽  
K 12 ◽  
Assembly Proteins

ABSTRACT CS1 pili are important virulence factors of enterotoxigenic Escherichia coli strains associated with human diarrheal disease. They are the prototype for a family of pili that share extensive sequence similarity among their structural and assembly proteins. Only four linked genes, cooB, cooA, cooC, and cooD, are required to produce CS1 pili in E. coli K-12. To identify amino acids important for the function of the major pilin CooA, we used alanine substitution mutagenesis targeting conserved residues in the N and C termini of the protein. To test function, we examined cooA mutants for the ability to agglutinate bovine erythrocytes. Each hemagglutination-negative (HA−) cooA mutant was examined to identify its assembly pathway defect. CooA has been shown to be degraded in the absence of CooB (K. Voegele, H. Sakellaris, and J. R. Scott, Proc. Natl. Acad. Sci. USA 94:13257-13261, 1997). We found several HA− cooA mutants that produced no detectable CooA, suggesting that recognition by CooB is mediated by residues in both the N and C termini of CooA. In addition, we found that alanine substitution for some of the conserved residues in the C-terminal motif “AGxYxG(x6)T,” which is found in all subunits of this pilus family, had no effect on pilus formation. However, alanine substitution for some of the alternating hydrophobic residues within this motif prevented CooA from interacting with CooD, which serves as both the tip adhesin and nucleation protein for pilus formation. Thus, it appears that some, but not all, of the residues in both the N and C termini of CooA play a critical role in the intermolecular interactions of the major pilin with the other structural and assembly proteins. We anticipate that the results obtained here for CS1 pili in enterotoxigenic E. coli will help develop an understanding of the pilus assembly pathway used by CS1 family members in several important human pathogens.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

DEVELOPMENT OF MULTIPLEX REAL-TIME PCR ASSAY FOR SIX PATHOGENS: RAPID TOOL FOR DIAGNOSIS OF BACTERIAL MENINGITIS

2019 ◽  
pp. 60-66
Author(s):  
Viet Quynh Tram Ngo ◽  
Thi Ti Na Nguyen ◽  
Hoang Bach Nguyen ◽  
Thi Tuyet Ngoc Tran ◽  
Thi Nam Lien Nguyen ◽  
...  
Keyword(s):  
Bacterial Meningitis ◽  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Methods ◽  
Type B ◽  
E Coli ◽  
Pcr Assays ◽  
Set Up ◽  
Etiological Agents ◽  
Suis Serotype

Introduction: Bacterial meningitis is an acute central nervous infection with high mortality or permanent neurological sequelae if remained undiagnosed. However, traditional diagnostic methods for bacterial meningitis pose challenge in prompt and precise identification of causative agents. Aims: The present study will therefore aim to set up in-house PCR assays for diagnosis of six pathogens causing the disease including H. influenzae type b, S. pneumoniae, N. meningitidis, S. suis serotype 2, E. coli and S. aureus. Methods: inhouse PCR assays for detecting six above-mentioned bacteria were optimized after specific pairs of primers and probes collected from the reliable literature resources and then were performed for cerebrospinal fluid (CSF) samples from patients with suspected meningitis in Hue Hospitals. Results: The set of four PCR assays was developed including a multiplex real-time PCR for S. suis serotype 2, H. influenzae type b and N. meningitides; three monoplex real-time PCRs for E. coli, S. aureus and S. pneumoniae. Application of the in-house PCRs for 116 CSF samples, the results indicated that 48 (39.7%) cases were positive with S. suis serotype 2; one case was positive with H. influenzae type b; 4 cases were positive with E. coli; pneumococcal meningitis were 19 (16.4%) cases, meningitis with S. aureus and N. meningitidis were not observed in any CSF samples in this study. Conclusion: our in-house real-time PCR assays are rapid, sensitive and specific tools for routine diagnosis to detect six mentioned above meningitis etiological agents. Key words: Bacterial meningitis, etiological agents, multiplex real-time PCR

scholarly journals Tailoring a Global Iron Regulon to a Uropathogen

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Rajdeep Banerjee ◽  
Erin Weisenhorn ◽  
Kevin J. Schwartz ◽  
Kevin S. Myers ◽  
Jeremy D. Glasner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Urinary Tract ◽  
Amino Acid ◽  
Regulatory Network ◽  
Iron Homeostasis ◽  
Iron Limitation ◽  
Content Type ◽  
E Coli ◽  
General Stress ◽  
K 12

ABSTRACT Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O2-dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O2 to produce aerobactin. IMPORTANCE Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes.

scholarly journals Knowledge extraction for assisted curation of summaries of bacterial transcription factor properties

Database ◽  
2020 ◽  
Vol 2020 ◽  
Author(s):  
Carlos-Francisco Méndez-Cruz ◽  
Antonio Blanchet ◽  
Alan Godínez ◽  
Ignacio Arroyo-Fernández ◽  
Socorro Gama-Castro ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Knowledge Extraction ◽  
Biomedical Literature ◽  
Main Role ◽  
E Coli ◽  
Bacterial Transcription ◽  
Manual Curation ◽  
New Knowledge ◽  
Serovar Typhimurium ◽  
K 12

Abstract Transcription factors (TFs) play a main role in transcriptional regulation of bacteria, as they regulate transcription of the genetic information encoded in DNA. Thus, the curation of the properties of these regulatory proteins is essential for a better understanding of transcriptional regulation. However, traditional manual curation of article collections to compile descriptions of TF properties takes significant time and effort due to the overwhelming amount of biomedical literature, which increases every day. The development of automatic approaches for knowledge extraction to assist curation is therefore critical. Here, we show an effective approach for knowledge extraction to assist curation of summaries describing bacterial TF properties based on an automatic text summarization strategy. We were able to recover automatically a median 77% of the knowledge contained in manual summaries describing properties of 177 TFs of Escherichia coli K-12 by processing 5961 scientific articles. For 71% of the TFs, our approach extracted new knowledge that can be used to expand manual descriptions. Furthermore, as we trained our predictive model with manual summaries of E. coli, we also generated summaries for 185 TFs of Salmonella enterica serovar Typhimurium from 3498 articles. According to the manual curation of 10 of these Salmonella typhimurium summaries, 96% of their sentences contained relevant knowledge. Our results demonstrate the feasibility to assist manual curation to expand manual summaries with new knowledge automatically extracted and to create new summaries of bacteria for which these curation efforts do not exist. Database URL: The automatic summaries of the TFs of E. coli and Salmonella and the automatic summarizer are available in GitHub (https://github.com/laigen-unam/tf-properties-summarizer.git).

scholarly journals Antagonism of Adherent Invasive E. coli LF82 With Human α-defensin 5 in the Follicle-associated Epithelium of Patients With Ileal Crohn’s Disease

2020 ◽  
Author(s):  
Lina Y Alkaissi ◽  
Martin E Winberg ◽  
Stéphanie DS Heil ◽  
Staffan Haapaniemi ◽  
Pär Myrelid ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Ex Vivo ◽  
Ussing Chambers ◽  
E Coli ◽  
Follicle Associated Epithelium ◽  
Vitro Model ◽  
Set Up

Abstract Background The first visible signs of Crohn’s disease (CD) are microscopic erosions over the follicle-associated epithelium (FAE). The aim of the study was to investigate the effects of human α-defensin 5 (HD5) on adherent-invasive Escherichia coli LF82 translocation and HD5 secretion after LF82 exposure in an in vitro model of human FAE and in human FAE ex vivo. Methods An in vitro FAE-model was set up by the coculture of Raji B cells and Caco-2-cl1 cells. Ileal FAE from patients with CD and controls were mounted in Ussing chambers. The effect of HD5 on LF82 translocation was studied by LF82 exposure to the cells or tissues with or without incubation with HD5. The HD5 secretion was measured in human FAE exposed to LF82 or Salmonella typhimurium. The HD5 levels were evaluated by immunofluorescence, immunoblotting, and ELISA. Results There was an increased LF82 translocation across the FAE-model compared with Caco-2-cl1 (P < 0.05). Incubation of cell/tissues with HD5 before LF82 exposure reduced bacterial passage in both models. Human FAE showed increased LF82 translocation in CD compared with controls and attenuated passage after incubation with sublethal HD5 in both CD and controls (P < 0.05). LF82 exposure resulted in a lower HD5 secretion in CD FAE compared with controls (P < 0.05), whereas Salmonella exposure caused equal secretion on CD and controls. There were significantly lower HD5 levels in CD tissues compared with controls. Conclusions Sublethal HD5 reduces the ability of LF82 to translocate through FAE. The HD5 is secreted less in CD in response to LF82, despite a normal response to Salmonella. This further implicates the integrated role of antimicrobial factors and barrier function in CD pathogenesis.

scholarly journals A genome-scale metabolic model of Saccharomyces cerevisiae that integrates expression constraints and reaction thermodynamics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Omid Oftadeh ◽  
Pierre Salvy ◽  
Maria Masid ◽  
Maxime Curvat ◽  
Ljubisa Miskovic ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Expression System ◽  
Biomass Composition ◽  
Industrial Biotechnology ◽  
Overflow Metabolism ◽  
Cellular Expression ◽  
A Genome ◽  
Wide Range ◽  
Eukaryotic Organisms ◽  
Genome Scale

AbstractEukaryotic organisms play an important role in industrial biotechnology, from the production of fuels and commodity chemicals to therapeutic proteins. To optimize these industrial systems, a mathematical approach can be used to integrate the description of multiple biological networks into a single model for cell analysis and engineering. One of the most accurate models of biological systems include Expression and Thermodynamics FLux (ETFL), which efficiently integrates RNA and protein synthesis with traditional genome-scale metabolic models. However, ETFL is so far only applicable for E. coli. To adapt this model for Saccharomyces cerevisiae, we developed yETFL, in which we augmented the original formulation with additional considerations for biomass composition, the compartmentalized cellular expression system, and the energetic costs of biological processes. We demonstrated the ability of yETFL to predict maximum growth rate, essential genes, and the phenotype of overflow metabolism. We envision that the presented formulation can be extended to a wide range of eukaryotic organisms to the benefit of academic and industrial research.

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