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Title: Growth Trade-Offs Accompany the Emergence of Glycolytic Metabolism in Shewanella oneidensis MR-1

Abstract

Bacteria increase their metabolic capacity via the acquisition of genetic material or by the mutation of genes already present in the genome. Here, we explore the mechanisms and trade-offs involved whenShewanella oneidensis, a bacterium that typically consumes small organic and amino acids, rapidly evolves to expand its metabolic capacity to catabolize glucose after a short period of adaptation to a glucose-rich environment. Using whole-genome sequencing and genetic approaches, we discovered that deletions in a region including the transcriptional repressor (nagR) that regulates the expression of genes associated with catabolism ofN-acetylglucosamine are the common basis for evolved glucose metabolism across populations. The loss ofnagRresults in the constitutive expression of genes for anN-acetylglucosamine permease (nagP) and kinase (nagK). We demonstrate that promiscuous activities of both NagP and NagK toward glucose allow for the transport and phosphorylation of glucose to glucose-6-phosphate, the initial events of glycolysis otherwise thought to be absent inS. oneidensis. 13C-based metabolic flux analysis uncovered that subsequent utilization was mediated by the Entner-Doudoroff pathway. This is an example whereby gene loss and preexisting enzymatic promiscuity, and not gain-of-function mutations, were the drivers of increased metabolic capacity. However, we observed a significant decrease in the growth rate on lactate after adaptationmore » to glucose catabolism, suggesting that trade-offs may explain why glycolytic function may not be readily observed inS. oneidensisin natural environments despite it being readily accessible through just a single mutational event.Gains in metabolic capacity are frequently associated with the acquisition of novel genetic material via natural or engineered horizontal gene transfer events. Here, we explored how a bacterium that typically consumes small organic acids and amino acids expands its metabolic capacity to include glucose via a loss of genetic material, a process frequently associated with a deterioration of metabolic function. Our findings highlight how the natural promiscuity of transporters and enzymes can be a key driver in expanding metabolic diversity and that many bacteria may possess a latent metabolic capacity accessible through one or a few mutations that remove regulatory functions. Our discovery of trade-offs between growth on lactate and on glucose suggests why this easily gained trait is not observed in nature.« less

Authors:
ORCiD logo [1];  [2]
  1. Univ. of Missouri, Columbia, MO (United States); Harvard Univ., Cambridge, MA (United States)
  2. Harvard Univ., Cambridge, MA (United States); Univ. of Idaho, Moscow, ID (United States)
Publication Date:
Research Org.:
Harvard Univ., Cambridge, MA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1425453
Grant/Contract Number:
SC0006739
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Journal of Bacteriology
Additional Journal Information:
Journal Volume: 199; Journal Issue: 11; Journal ID: ISSN 0021-9193
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Chubiz, Lon M., and Marx, Christopher J.. Growth Trade-Offs Accompany the Emergence of Glycolytic Metabolism in Shewanella oneidensis MR-1. United States: N. p., 2017. Web. doi:10.1128/JB.00827-16.
Chubiz, Lon M., & Marx, Christopher J.. Growth Trade-Offs Accompany the Emergence of Glycolytic Metabolism in Shewanella oneidensis MR-1. United States. doi:10.1128/JB.00827-16.
Chubiz, Lon M., and Marx, Christopher J.. Mon . "Growth Trade-Offs Accompany the Emergence of Glycolytic Metabolism in Shewanella oneidensis MR-1". United States. doi:10.1128/JB.00827-16. https://www.osti.gov/servlets/purl/1425453.
@article{osti_1425453,
title = {Growth Trade-Offs Accompany the Emergence of Glycolytic Metabolism in Shewanella oneidensis MR-1},
author = {Chubiz, Lon M. and Marx, Christopher J.},
abstractNote = {Bacteria increase their metabolic capacity via the acquisition of genetic material or by the mutation of genes already present in the genome. Here, we explore the mechanisms and trade-offs involved whenShewanella oneidensis, a bacterium that typically consumes small organic and amino acids, rapidly evolves to expand its metabolic capacity to catabolize glucose after a short period of adaptation to a glucose-rich environment. Using whole-genome sequencing and genetic approaches, we discovered that deletions in a region including the transcriptional repressor (nagR) that regulates the expression of genes associated with catabolism ofN-acetylglucosamine are the common basis for evolved glucose metabolism across populations. The loss ofnagRresults in the constitutive expression of genes for anN-acetylglucosamine permease (nagP) and kinase (nagK). We demonstrate that promiscuous activities of both NagP and NagK toward glucose allow for the transport and phosphorylation of glucose to glucose-6-phosphate, the initial events of glycolysis otherwise thought to be absent inS. oneidensis.13C-based metabolic flux analysis uncovered that subsequent utilization was mediated by the Entner-Doudoroff pathway. This is an example whereby gene loss and preexisting enzymatic promiscuity, and not gain-of-function mutations, were the drivers of increased metabolic capacity. However, we observed a significant decrease in the growth rate on lactate after adaptation to glucose catabolism, suggesting that trade-offs may explain why glycolytic function may not be readily observed inS. oneidensisin natural environments despite it being readily accessible through just a single mutational event.Gains in metabolic capacity are frequently associated with the acquisition of novel genetic material via natural or engineered horizontal gene transfer events. Here, we explored how a bacterium that typically consumes small organic acids and amino acids expands its metabolic capacity to include glucose via a loss of genetic material, a process frequently associated with a deterioration of metabolic function. Our findings highlight how the natural promiscuity of transporters and enzymes can be a key driver in expanding metabolic diversity and that many bacteria may possess a latent metabolic capacity accessible through one or a few mutations that remove regulatory functions. Our discovery of trade-offs between growth on lactate and on glucose suggests why this easily gained trait is not observed in nature.},
doi = {10.1128/JB.00827-16},
journal = {Journal of Bacteriology},
number = 11,
volume = 199,
place = {United States},
year = {Mon Mar 13 00:00:00 EDT 2017},
month = {Mon Mar 13 00:00:00 EDT 2017}
}

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  • Biofilms are known to possess spatially and temporally varying metabolite concentration profiles at the macroscopic and microscopic scales. This results in varying growth environments within that may ultimately drive species diversity, determine biofilm structure and also the spatial arrangement of the community members. Using noninvasive nuclear magnetic resonance (NMR) microscopic imaging/spectroscopy and confocal imaging, we investigated anaerobic reduction kinetics, structural variation, and the stratification of metabolism within live biofilms of the facultative anaerobic dissimilatory metal-reducing Shewanella oneidensis strain MR-1. Biofilms were pregrown using a defined minimal media in a homebuilt constant depth film fermenter and subsequently transferred to an in-magnetmore » sample chamber under laminar flow for NMR measurements. The sample was subjected to various, rapidly switched substrate/ anaerobic electron acceptor combinations (fumarate, dimethyl sulfoxide, and nitrate electron acceptors). Localized NMR spectroscopy was used to non-invasively monitored the spectra of hydrogen-containing metabolites at high temporal resolution (4.5 min) under oxygen-limited conditions. Anaerobic reduction was immediately observed upon switching feed solutions indicate that no gene induction (transcriptional response) was needed for MR-1 to switch between fumarate, dimethyl sulfoxide (DMSO) and nitrate electron acceptors. In parallel experiments, confocal microscopy was used with constitutively expressed fluorescent reporters to independently investigate structural changes in response to the availability of electron acceptor and also the outcome of metabolic competition under oxygen-limited conditions. A clearer understanding of the metabolic diversity and plasticity of the biofilm mode of growth as well as how this possibly translates to the environmental fitness is made possible through the use of non-invasive and non-destructive techniques such as described here.« less
  • The ability to utilize lactate as a sole source of carbon and energy is one of the key metabolic signatures of Shewanellae, a diverse group of dissimilatory metal reducing bacteria commonly found in aquatic and sedimentary environments. Nonetheless, homology searches failed to recognize orthologs of previously described bacterial D- or L-lactate oxidizing enzymes (Escherichia coli genes dld and lldD) in any of the 13 analyzed genomes of Shewanella spp. Using comparative genomic techniques, we identified a conserved chromosomal gene cluster in Shewanella oneidensis MR-1 (locus tag: SO1522-SO1518) containing lactate permease and candidate genes for both D- and L-lactate dehydrogenase enzymes.more » The predicted D-LDH gene (dldD, SO1521) is a distant homolog of FAD-dependent lactate dehydrogenase from yeast, whereas the predicted L-LDH is encoded by three genes with previously unknown functions (lldEGF, SO1520-19-18). Through a combination of genetic and biochemical techniques, we experimentally confirmed the predicted physiological role of these novel genes in S. oneidensis MR-1 and carried out successful functional validation studies in Escherichia coli and Bacillus subtilis. We conclusively showed that dldD and lldEFG encode fully functional D-and L-LDH enzymes, which catalyze the oxidation of the respective lactate stereoisomers to pyruvate. Notably, the S. oneidensis MR-1 LldEFG enzyme is the first described example of a multi-subunit lactate oxidase. Comparative analysis of >400 bacterial species revealed the presence of LldEFG and Dld in a broad range of diverse species accentuating the potential importance of these previously unknown proteins in microbial metabolism.« less
  • Shewanellae are gram-negative facultatively anaerobic metal-reducing bacteria commonly found in chemically (i.e., redox) stratified environments. Occupying such niches requires the ability to rapidly acclimate to changes in electron donor/acceptor type and availability; hence, the ability to compete and thrive in such environments must ultimately be reflected in the organization and flexibility of the electron transfer networks as well as central and peripheral carbon metabolism pathways. To understand the factors contributing to the ecophysiological success of Shewanellae, the metabolic network of S. oneidensis MR-1 was reconstructed. The resulting network consists of 774 reactions, 783 genes, and 634 unique metabolites and containsmore » biosynthesis pathways for all cell constituents. Using constraint-based modeling, we investigated aerobic growth of S. oneidensis MR-1 on numerous carbon sources. To achieve this, we (i) used experimental data to formulate a biomass equation and estimate cellular ATP requirements, (ii) developed an approach to identify futile cycles, (iii) classified how reaction usage affects cellular growth, (iv) predicted cellular biomass yields on different carbon sources and compared model predictions to experimental measurements, and (v) used experimental results to refine metabolic fluxes for growth on lactate. The results revealed that aerobic lactate-grown cells of S. oneidensis MR-1 used less efficient enzymes to couple electron transport to proton motive force generation, and possibly operated at least one futile cycle involving malic enzymes. Several examples are provided whereby model predictions were validated by experimental data, in particular the role of serine hydroxymethyltransferase and glycine cleavage system in the metabolism of one-carbon units, and growth on different sources of carbon and energy. This work illustrates how integration of computational and experimental efforts facilitates the understanding of microbial metabolism at a systems level and the discovery of new physiological properties.« less
  • The role of LuxS in Shewanella oneidensis MR-1 has been examined by transcriptomic profiling, biochemical, and physiological experiments. The results indicate that a mutation in luxS alters biofilm development, not by altering quorum-sensing abilities but by disrupting the activated methyl cycle (AMC). The S. oneidensis wild type can produce a luminescence response in the AI-2 reporter strain Vibrio harveyi MM32. This luminescence response is abolished upon the deletion of luxS. The deletion of luxS also alters biofilm formations in static and flowthrough conditions. Genetic complementation restores the mutant biofilm defect, but the addition of synthetic AI-2 has no effect. Thesemore » results suggest that AI-2 is not used as a quorum-sensing signal to regulate biofilm development in S. oneidensis. Growth on various sulfur sources was examined because of the involvement of LuxS in the AMC. A mutation in luxS produced a reduced ability to grow with methionine as the sole sulfur source. Methionine is a key metabolite used in the AMC to produce a methyl source in the cell and to recycle homocysteine. These data suggest that LuxS is important to metabolizing methionine and the AMC in S. oneidensis.« less
  • EtrA in Shewanella oneidensis MR-1, a model organism for study of adaptation to varied redox niches, shares 73.6% and 50.8% amino acid sequence identity with the oxygen-sensing regulators Fnr in E. coli and Anr in Pseudomonas aeruginosa, respectively; however, its regulatory role of anaerobic metabolism in Shewanella spp. is not well understood. The expression of the nap genes, nrfA, cymA and hcp was significantly reduced in etrA deletion mutant EtrA7-1; however, limited anaerobic growth and nitrate reduction occurred, suggesting that multiple regulators control nitrate reduction in this strain. Dimethyl sulfoxide (DMSO) and fumarate reductase gene expression was down regulated atmore » least 2-fold and the EtrA7-1 mutant grew poorly with fumarate and dimethyl sulfoxide (DMSO), suggesting both respiratory pathways are under EtrA control. Transcript analysis further suggested a role of EtrA in prophage activation and down regulation of genes implicated in aerobic metabolism. In contrast to previous studies that attributed a minor regulatory role to EtrA in Shewanella spp., this study demonstrates that EtrA acts as a global transcriptional regulator and confers physiological advantages to strain MR-1 under certain growth conditions. In conjunction with other regulators, EtrA fine-tunes the expression of genes involved in anaerobic metabolism in S. oneidensis strain MR-1.« less