scholarly journals Nanotube-mediated cross-feeding couples the metabolism of interacting bacterial cells

2017 ◽  
Author(s):  
Shraddha Shitut ◽  
Tobias Ahsendorf ◽  
Samay Pande ◽  
Matthew Egbert ◽  
Christian Kost
Keyword(s):  
Amino Acid ◽  
Feedback Inhibition ◽  
Bacterial Cells ◽  
Loss Of Function ◽  
E Coli ◽  
Donor Cells ◽  
Mere Presence ◽  
Source Sink ◽  
Amino Acid Concentrations ◽  
Amino Acid Biosynthetic Pathway

ABSTRACTBacteria frequently engage in cross-feeding interactions that involve an exchange of metabolites with other micro- or macroorganisms. The often obligate nature of these associations, however, hampers manipulative experiments, thus limiting our mechanistic understanding of the ecophysiological consequences that result for the organisms involved. Here we address this issue by taking advantage of a well-characterised experimental model system, in which auxotrophic genotypes ofE. coliderive essential amino acid from prototrophic donor cells using intercellular nanotubes. Surprisingly, donor-recipient cocultures revealed that the mere presence of auxotrophic genotypes in coculture was sufficient to increase amino acid production levels in donor cells. Subsequent experiments unravelled that this effect was due to the depletion of amino acid concentrations in the cytoplasm of donor cells, which delayed feedback inhibition of the corresponding amino acid biosynthetic pathway. This finding indicates that in newly established mutualistic associations, an intercellular regulation of exchanged metabolites can simply emerge from the architecture of the underlying biosynthetic pathways, rather than through the evolution of new regulatory mechanisms. Taken together, our results show that a single loss-of-function mutation can physiologically couple the metabolism of two cross-feeding cells in a source-sink-like relationship.

Cellular Amino Acid Concentrations and Regulation of Aspartate Kinase in the Thermophilic Phototrophic Prokaryote Chloroflexus aurantiacus

1990 ◽  
Vol 45 (1-2) ◽  
pp. 74-78 ◽  
Author(s):  
Jobst-Heinrich Klemme ◽  
Gisela Laakmann-Ditges ◽  
Jutta Mertschuweit
Keyword(s):  
Amino Acid ◽  
Feedback Inhibition ◽  
Gel Filtration ◽  
Break Point ◽  
Test System ◽  
Chloroflexus Aurantiacus ◽  
Aspartate Kinase ◽  
Homoserine Dehydrogenase ◽  
Molecular Weights ◽  
Amino Acid Concentrations

Aspartate kinase (AK , EC 2.7.2.4) from the thermophilic, phototrophic prokaryote, Chloroflexus aurantiacus, was partially purified and separated from homoserine dehydrogenase (HSDH, EC 1.1.1.3). The molecular weights as determined by gel filtration were 130,000 and 46,000, respectively. HSDH had a moderately high thermal stability (50% inactivation at 84 °C) and displayed its activity optimum at 72 °C. By contrast, AK had its activity optimum at 52 °C (with a break-point in the Arrhenius plot at 42 °C) and was much less thermostable (50% inactivation at 67 °C). The Km-values for aspartate and ATP (determined in a pyruvate kinase-coupled test system) were 10.5 and 0.63 mM , respectively. The enzyme was strongly inhibited by L-threonine (Ki = 10 μm) and activated by alanine, isoleucine, valine and methionine. L-Threonine acted as a mixed-type inhibitor in respect to aspartate, and non-competitively in respect to ATP. Contrary to AKs from Rhodospirillaceae, the enzyme from Chloroflexus aurantiacus was not subject to a concerted feedback inhibition by two amino acids of the aspartate family. The regulatory properties of the aspartate kinase are discussed in relation to the cellular amino acid concentrations.

scholarly journals The Chemical Chaperone Proline Relieves the Thermosensitivity of a dnaK Deletion Mutant at 42°C

2004 ◽  
Vol 186 (23) ◽  
pp. 8149-8152 ◽  
Author(s):  
Madhab K. Chattopadhyay ◽  
Renée Kern ◽  
Michel-Yves Mistou ◽  
Abhaya M. Dandekar ◽  
Sandra L. Uratsu ◽  
...  
Keyword(s):  
Protein Aggregation ◽  
Citrate Synthase ◽  
Feedback Inhibition ◽  
Single Mutation ◽  
Two Dimensional Gel Electrophoresis ◽  
Chemical Chaperone ◽  
E Coli ◽  
Amino Acid Biosynthetic Pathway

ABSTRACT Since, like other osmolytes, proline can act as a protein stabilizer, we investigated the thermoprotectant properties of proline in vitro and in vivo. In vivo, elevated proline pools in Escherichia coli (obtained by altering the feedback inhibition by proline of γ-glutamylkinase, the first enzyme of the proline biosynthesis pathway) restore the viability of a dnaK-deficient mutant at 42°C, suggesting that proline can act as a thermoprotectant for E. coli cells. Furthermore, analysis of aggregated proteins in the dnaK-deficient strain at 42°C by two-dimensional gel electrophoresis shows that high proline pools reduce the protein aggregation defect of the dnaK-deficient strain. In vitro, like other “chemical chaperones,” and like the DnaK chaperone, proline protects citrate synthase against thermodenaturation and stimulates citrate synthase renaturation after urea denaturation. These results show that a protein aggregation defect can be compensated for by a single mutation in an amino acid biosynthetic pathway and that an ubiquitously producible chemical chaperone can compensate for a defect in one of the major chaperones involved in protein folding and aggregation.

scholarly journals Amino acid deprivation and its effect on mating ability inEscherichia coliK12

1966 ◽  
Vol 8 (1) ◽  
pp. 115-118 ◽  
Author(s):  
K. W. Fisher
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Deprivation ◽  
Mating Ability ◽  
Chromosome Transfer ◽  
E Coli ◽  
Donor Cells ◽  
Relationship Of ◽  
The Relationship

The conclusion by Suit, Matney, Doudney & Billen (1964) that Hfr donor cells ofEscherichia coliK12, starved of required amino acids can mate, has been re-examined. It appears that their conclusion is not valid and that apparent fertility of amino-acid starved cells is due to cross-feeding by the F−cells. The relationship of this result to the alternative mechanisms for chromosome transfer inE. coliis discussed.

scholarly journals Allosteric Feedback Inhibition Enables Robust Amino Acid Biosynthesis in E. coli by Enforcing Enzyme Overabundance

Cell Systems ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 66-75.e8 ◽  
Author(s):  
Timur Sander ◽  
Niklas Farke ◽  
Christoph Diehl ◽  
Michelle Kuntz ◽  
Timo Glatter ◽  
...  
Keyword(s):  
Amino Acid ◽  
Feedback Inhibition ◽  
Amino Acid Biosynthesis ◽  
Acid Biosynthesis ◽  
E Coli

scholarly journals Characterization of the Avian PathogenicEscherichia coli Hemagglutinin Tsh, a Member of the Immunoglobulin A Protease-Type Family of Autotransporters

1999 ◽  
Vol 67 (2) ◽  
pp. 772-781 ◽  
Author(s):  
Christos Stathopoulos ◽  
David L. Provence ◽  
Roy Curtiss
Keyword(s):  
Amino Acid ◽  
Sequence Data ◽  
Single Gene ◽  
Shigella Flexneri ◽  
Immunoglobulin A ◽  
Site Directed Mutagenesis ◽  
Bacterial Cells ◽  
E Coli ◽  
K 12

ABSTRACT We reported earlier that a single gene, tsh, isolated from a strain of avian pathogenic Escherichia coli (APEC) was sufficient to confer on E. coli K-12 a hemagglutinin-positive phenotype and that the deduced sequence of the Tsh protein shared homology to the serine-type immunoglobulin A (IgA) proteases of Neisseria gonorrhoeae and Haemophilus influenzae. In this report we show that E. coli K-12 containing the recombinant tsh gene produced two proteins, a 106-kDa extracellular protein and a 33-kDa outer membrane protein, and was also able to agglutinate chicken erythrocytes. N-terminal sequence data indicated that the 106-kDa protein, designated Tshs, was derived from the N-terminal end of Tsh after the removal of a 52-amino-acid N-terminal signal peptide, while the 33-kDa protein, designated Tshβ, was derived from the C-terminal end of Tsh starting at residue N1101. The Tshsdomain contains the 7-amino-acid serine protease motif that includes the active-site serine (S259), found also in the secreted domains of the IgA proteases. However, site-directed mutagenesis of S259 did not abolish the hemagglutinin activity or the extracellular secretion of Tshs indicating that host-directed proteolysis was mediating the release of Tshs. Studies with an E. coli K-12ompT mutant strain showed that the surface protease OmpT was not needed for the secretion of Tshs. Tsh belongs to a subclass of the IgA protease family, which also includes EspC of enteropathogenic E. coli, EspP of enterohemorragic E. coli, and SepA and VirG of Shigella flexneri, which seem to involve a host endopeptidase to achieve extracellular release of their N-terminal domains. In proteolytic studies conducted in vitro, Tshs did not cleave the substrate of the IgA proteases, human IgA1 or chicken IgA, and did not show proteolytic activity in a casein-based assay. Correlation of Tsh expression and hemagglutination activity appears to be a very complex phenomenon, influenced by strain and environmental conditions. Nevertheless, for both APEC and recombinant E. coli K-12 strains containing thetsh gene, it was only the whole bacterial cells and not the cell-free supernatants that could confer hemagglutinin activity. Our results provide insights into the expression, secretion, and proteolytic features of the Tsh protein, which belongs to the growing family of gram-negative bacterial extracellular virulence factors, named autotransporters, which utilize a self-mediated mechanism to achieve export across the bacterial cell envelope.

Carbon and nitrogen balance in beech roots under competitive pressure of soil-borne microorganisms induced by girdling, drought and glucose application

2010 ◽  
Vol 37 (9) ◽  
pp. 879 ◽  
Author(s):  
Jana B. Winkler ◽  
Michael Dannenmann ◽  
Judy Simon ◽  
Rodica Pena ◽  
Christine Offermann ◽  
...  
Keyword(s):  
Amino Acid ◽  
Feedback Inhibition ◽  
Root Tip ◽  
Pulse Labelling ◽  
Root Tips ◽  
Free Living ◽  
Soil System ◽  
Carbon And Nitrogen ◽  
Fagus Sylvatica L ◽  
Amino Acid Concentrations

The goal of this work was to increase the understanding of factors regulating nitrogen (N) competition between roots and soil microbes. For this purpose, root assimilate supply was diminished or abolished in beech (Fagus sylvatica L.) seedlings by girdling, drought stress or a combination of both factors. This was revealed by 13C tracer abundance in root tips after 13CO2 pulse labelling of the shoots. Analysis of different root tip fractions revealed that only 6% were ectomycorrhizal. Carbon (C) allocation to ectomycorrhizal and vital non-mycorrhizal root tips was ~26% higher than to distorted root tips. Drought resulted in ~30% increased ammonium (NH4+) and amino acid concentrations in roots and ~65% increased soil NH4+ concentrations, probably because of lower consumption of NH4+ by free-living microorganisms. Root uptake of glutamine of 13 nmol g–1 fresh mass h–1 decreased 2-fold with drought, although the number of vital root tips did not decrease. Carbon content in biomass of free-living microbes increased with glucose application regardless of drought, resulting in significant depletion in soil nitrate (NO3–), root NH4+ and amino acid concentrations. Our results suggest that the root–soil system of young beech trees was C-limited, and this prevented amino acid metabolism in roots and microbial NO3– consumption in the soil, thereby exerting feedback inhibition on uptake of inorganic N by roots. We suggest that rhizodeposition is a key link in regulating the plant–microbial N balance.

scholarly journals Effect of Drug Transporter Genes on Cysteine Export and Overproduction in Escherichia coli

2006 ◽  
Vol 72 (7) ◽  
pp. 4735-4742 ◽  
Author(s):  
Satoshi Yamada ◽  
Naoki Awano ◽  
Kyoko Inubushi ◽  
Eri Maeda ◽  
Shigeru Nakamori ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Feedback Inhibition ◽  
Tetracycline Resistance ◽  
Industrial Applications ◽  
Genetically Engineered ◽  
Drug Transporter ◽  
E Coli ◽  
Important Amino Acid ◽  
Gene Assay

ABSTRACT l-Cysteine is an important amino acid in terms of its industrial applications. We previously found a marked production of l-cysteine from glucose in recombinant Escherichia coli cells expressing an altered cysE gene encoding feedback inhibition-insensitive serine acetyltransferase. Also, a lower level of cysteine desulfhydrase (CD) activity, which is involved in l-cysteine degradation, increased l-cysteine productivity in E. coli. The use of an l-cysteine efflux system could be promising for breeding l-cysteine overproducers. In addition to YdeD and YfiK, which have been reported previously as l-cysteine exporter proteins in E. coli, we analyzed the effects of 33 putative drug transporter genes in E. coli on l-cysteine export and overproduction. Overexpression of the acrD, acrEF, bcr, cusA, emrAB, emrKY, ybjYZ, and yojIH genes reversed the growth inhibition of tnaA (the major CD gene)-disrupted E. coli cells by l-cysteine. We also found that overexpression of these eight genes reduces intracellular l-cysteine levels after cultivation in the presence of l-cysteine. Amino acid transport assays showed that Bcr overexpression conferring bicyclomycin and tetracycline resistance specifically promotes l-cysteine export driven by energy derived from the proton gradient. When a tnaA-disrupted E. coli strain expressing the altered cysE gene was transformed with a plasmid carrying the bcr gene, the transformant exhibited more l-cysteine production than cells carrying the vector only. A reporter gene assay suggested that the bcr gene is constitutively expressed at a substantial level. These results indicate that the multidrug transporter Bcr in the major facilitator family is involved in l-cysteine export and overproduction in genetically engineered E. coli cells.

scholarly journals The Hek Outer Membrane Protein of Escherichia coli Strain RS218 Binds to Proteoglycan and Utilizes a Single Extracellular Loop for Adherence, Invasion, and Autoaggregation

2007 ◽  
Vol 76 (3) ◽  
pp. 1135-1142 ◽  
Author(s):  
Robert P. Fagan ◽  
Matthew A. Lambert ◽  
Stephen G. J. Smith
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Epithelial Cells ◽  
Membrane Protein ◽  
Molecular Level ◽  
Coli Strain ◽  
Bacterial Cells ◽  
Extracellular Loop ◽  
Gram Negative ◽  
E Coli

ABSTRACT Escherichia coli is the principal gram-negative causative agent of sepsis and meningitis in neonates. The pathogenesis of meningitis due to E. coli K1 involves mucosal colonization, transcytosis of epithelial cells, survival in the bloodstream, and eventually invasion of the meninges. The last two aspects have been well characterized at a molecular level. Less is known about the early stages of pathogenesis, i.e., adhesion to and invasion of epithelial cells. We have previously reported that the Hek protein causes autoaggregation and can mediate adherence to and invasion of epithelial cells. Here, we report that Hek-mediated adherence is dependent on binding to glycosoaminoglycan, in particular, heparin. The ability to hemagglutinate, autoaggregate, adhere, and invade is contingent on a putative 25-amino-acid loop that is exposed to the outside of the bacterial cells.

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