Large-Scale 13C Flux Profiling Reveals Conservation of the Entner-Doudoroff Pathway as a Glycolytic Strategy among Marine BacteriaThat Use Glucose

Arne Klingner,a Annekathrin Bartsch,a Marco Dogs,b Irene Wagner-Döbler,c Dieter Jahn,d Meinhard Simon,b Thorsten Brinkhoff,b

Judith Becker,e Christoph Wittmanne

Institute of Biochemical Engineering, Technical University Braunschweig, Braunschweig, Germanya; Institute for Chemistry and Biology of the Marine Environment,University of Oldenburg, Oldenburg, Germanyb; Helmholtz Centre for Infection Research, Research Group Microbial Communication, Braunschweig, Germanyc; Instituteof Microbiology, Technical University Braunschweig, Braunschweig, Germanyd; Institute of Systems Biotechnology, Saarland University, Saarbrücken, Germanye

Marine bacteria form one of the largest living surfaces on Earth, and their metabolic activity is of fundamental importance forglobal nutrient cycling. Here, we explored the largely unknown intracellular pathways in 25 microbes representing differentclasses of marine bacteria that use glucose: Alphaproteobacteria, Gammaproteobacteria, and Flavobacteriia of the Bacteriodetesphylum. We used 13C isotope experiments to infer metabolic fluxes through their carbon core pathways. Notably, 90% of allstrains studied use the Entner-Doudoroff (ED) pathway for glucose catabolism, whereas only 10% rely on the Embden-Meyer-hof-Parnas (EMP) pathway. This result differed dramatically from the terrestrial model strains studied, which preferentiallyused the EMP pathway yielding high levels of ATP. Strains using the ED pathway exhibited a more robust resistance against theoxidative stress typically found in this environment. An important feature contributing to the preferential use of the ED pathwayin the oceans could therefore be enhanced supply of NADPH through this pathway. The marine bacteria studied did not specifi-cally rely on a distinct anaplerotic route, but the carboxylation of phosphoenolpyruvate (PEP) or pyruvate for fueling of the tri-carboxylic acid (TCA) cycle was evenly distributed. The marine isolates studied belong to clades that dominate the uptake of glu-cose, a major carbon source for bacteria in seawater. Therefore, the ED pathway may play a significant role in the cycling ofmono- and polysaccharides by bacterial communities in marine ecosystems.

Marine bacteria influence global environmental dynamics infundamental ways by controlling the biogeochemistry and

productivity of the oceans (1). Due to their importance, marinemicroorganisms have been studied intensively (2). In particular,their mechanisms for metabolizing carbon and other nutrientshave attracted attention, because they directly or indirectly affectthe biogeochemical status of seawater (3). A prominent nutrientin seawater is glucose, the most abundant free neutral aldose (4).Current estimates of glucose concentrations in seawater indicatean almost ubiquitous distribution in the oceans in a nanomolarrange (5). Particularly, large amounts of glucose are available incoastal habitats, e.g., during bloom situations (6). In fact, a largefraction (�30%) of bacterial growth can be supported by thismonosaccharide in some oceans (7, 8). Furthermore, glucose isthe dominant component of dissolved polysaccharides, whichconstitute up to 15% of marine dissolved organic matter (9). Theturnover of the (monomeric and polymeric) glucose pool in dif-ferent oceanic regions ranges from days to months, and glucoseassimilation in marine surface waters may represent up to 40% ofbacterial carbon production (5). Taken together, bacteria that useglucose are common in the sea (10), and glucose is a representativemodel nutrient to monitor carbon uptake by heterotrophic ma-rine bacteria (11). At this point, questions that arise from currentknowledge concern the intracellular pathways involved in glucoseutilization. Generally, three common alternative routes occur inbacteria for catabolic breakdown of glucose, the Embden-Meyer-hof-Parnas (EMP) pathway (EMPP), Entner-Doudoroff (ED)pathway (EDP), and pentose phosphate (PP) pathway (PPP) (12).The EMP pathway is nearly ubiquitous in the bacterial kingdom(13). It has been argued that this relates to the superior energy

efficiency of the EMP pathway (equation 1): it yields twice asmuch ATP as the ED pathway (equation 2) (14).

Glucose � 2 ADP � 2 NAD� → 2 pyruvate � 2 ATP

� 2 NADH (1)

Glucose � ADP � NAD� � NADP� → 2 pyruvate � ATP

� NADH � NADPH (2)

However, phylogenetically distinct bacteria (Firmicutes, Alp-haproteobacteria, and Gammaproteobacteria), including aerobesand anaerobes, autotrophs and heterotrophs, rely on the ED path-way for glucose catabolism (15, 16). Quantitative flux through theEMP, ED, and PP pathways is accessible via 13C fluxomics; in theseexperiments, quantitative information on fluxes, i.e., the in vivo

Received 26 September 2014 Accepted 19 January 2015

Accepted manuscript posted online 23 January 2015

Citation Klingner A, Bartsch A, Dogs M, Wagner-Döbler I, Jahn D, Simon M,Brinkhoff T, Becker J, Wittmann C. 2015. Large-scale 13C flux profiling revealsconservation of the Entner-Doudoroff pathway as a glycolytic strategy amongmarine bacteria that use glucose. Appl Environ Microbiol 81:2408 –2422.doi:10.1128/AEM.03157-14.

Editor: H. Nojiri

Address correspondence to Christoph Wittmann,christoph.wittmann@uni-saarland.de.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.03157-14.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.03157-14

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activities of intracellular enzymes and pathways, is obtained fromthe labeling patterns of metabolites generated during growth onspecific 13C tracer substrates (17, 18). Such studies have providedvaluable insight into the intracellular pathways of microorgan-isms. For example, industrially relevant microbes have been ex-tensively studied (19), and more recently, fluxomic studies haveexamined pathogenic (16, 20) and ecologically important (21)microorganisms. In contrast, there is still limited knowledge onthe intracellular pathways of marine bacteria, and only a few iso-lates have been investigated so far (22–24). A more comprehensiveanalysis of intracellular pathways in marine microorganismspromises a better understanding of the ways they survive in thehighly heterogeneous oceans (25) and their susceptibility to envi-ronmental variability and climate change (2). Here, we studied thecentral carbon pathways of marine microbes that use glucose onthe level of metabolic fluxes. We selected 25 strains of bacteriabelonging to various classes of the Bacteriodetes phylum, Alpha-proteobacteria, Gammaproteobacteria, and Flavobacteriia, as rep-resentative and ubiquitous classes of marine bacterial communi-ties (26). Strains of Actinobacteria that are indicators forfreshwater input were also included (27). We quantified metabolicfluxes in cells grown in a seawater medium with glucose as a modelsubstrate. In addition to the major catabolic pathways, i.e., theEMP, ED, and PP pathways, we analyzed fluxes through anaple-rotic pathways that are important for biosynthesis and fueling. Weintegrated the flux data obtained with data on the enzymatic in-ventories and on oxidative stress tolerance to generate a carefullycurated conceptual representation of the metabolic strategies thatpermit microbes to grow and thrive in the oceans.

MATERIALS AND METHODSBacterial strains. We studied a set of marine model strains and severalterrestrial model strains (see Fig. 1 and Table 1; also see Table S1 in thesupplemental material). The sequenced marine-type strains Dinoroseo-bacter shibae (28), Alteromonas macleodii (29), Eudoraea adriatica (30),Polaribacter dokdonensis (31), Pseudoalteromonas haloplanktis (32), Pseu-doalteromonas marina (33), and Phaeobacter inhibens DSM 17395 (34),which is not the type strain, were obtained from culture collections (TableS1). The marine Pseudoalteromonas sp. strains HEL-36, HEL-40, andHEL-49, Alteromonas sp. strains HEL-5, HEL-51, BIO-267, and BIO-296,and Leeuwenhoekiella sp. strain Pic90 were previously isolated from watersamples and biofilms near the island of Helgoland, Germany, in the NorthSea and phylogenetically classified using 16S rRNA gene sequencing (35).Phyllobacteriaceae bacterium TK, Oceanospirillaceae bacterium T17,Pseudonocardiaceae bacterium T4, Flavobacteriaceae bacterium TN, Fla-vobacteriaceae bacterium T15 (36), as well as Pseudomonas sp. strainGWS-TZ-H209, Vibrio sp. strain GWS-TZ-H304, and Gammaproteobac-teriaceae bacterium GWS-SE-H233 (37) originated from an intertidalmudflat region of the southern North Sea (Table S1). Jannaschia sp. strainB3 and Loktanella sp. strain D3 were isolated from the surface of themarine macroalga Fucus spiralis, collected in June 2010 from the intertidalflat area in Neuharlingersiel on the North Sea coast of Germany. Thesamples were transported on ice to the laboratory in seawater collectedon-site. The alga was washed three times with sterile-filtered and auto-claved seawater to remove unattached bacteria and particles, spread on aplate with marine agar (marine agar 2216; Becton Dickinson, FranklinLakes, NJ, USA), and then incubated for 2 weeks at 25°C in the dark. Singlecolonies were selected and transferred at least three times to isolate purestrains. The purity of the strains in culture and in the experiments wastested using denaturing gradient gel electrophoresis (38). The 16S rRNAgenes of the two strains were amplified and sequenced (39). For phyloge-netic analysis, sequence reads of at least 650 bp were compared withGenBank entries using the BLAST analysis tool of the National Center for

Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov). For com-parison, nonmarine Escherichia coli K-12 (ATCC 10798), Bacillus subtilis168 (DSM 402), Bacillus megaterium (DSM 319), Corynebacterium glu-tamicum (ATCC 13032), and Pseudomonas putida KT 2440 (DSM 6125)were obtained from the corresponding culture collections. All strains weremaintained as glycerol stocks at �70°C.

Media. For growth on agar plates, 37.4 g liter�1 of marine broth (ma-rine broth 2216; Becton Dickinson) was mixed with 15 g liter�1 of agar(Becton Dickinson). In addition, we used marine broth without agar as aliquid medium for the first precultures. A defined medium was used forthe second precultures and the main cultures (40). This medium con-tained the following (per liter): 1.8 g of glucose, 4.0 g of Na2SO4, 0.2 g ofKH2PO4, 0.25 g of NH4Cl, 20.0 g of NaCl, 9.0 g of MgCl2·6H2O, 0.5 g ofKCl, 0.15 g of CaCl2·2H2O, 0.19 g of NaHCO3, 4.2 mg of FeSO4·7H2O,10.4 mg of Titriplex-(III) (Na2-EDTA), 60 �g of H3BO3, 200 �g ofMnCl2·4H2O, 380 �g of CoCl2·6H2O, 48 �g of NiCl2·6H2O, 4 �g ofCuCl2·2H2O, 288 �g of ZnSO4·7H2O, and 72 �g of Na2MoO4·2H2O.Trace elements were added to the autoclaved basal medium from a sterile-filtered 500� stock solution. The pH of the final medium was adjusted to8.0 using 2 M NaOH. The medium was supplemented with 5 mg liter�1

4-aminobenzoic acid, 2 mg liter�1 folic acid, 2 mg liter�1 biotin, 5 mgliter�1 nicotinic acid, 5 mg liter�1 Ca-pantothenic acid, 5 mg liter�1 vi-tamin B2, 10 mg liter�1 vitamin B6, 0.1 mg liter�1 vitamin B12, 5 mgliter�1 thiamine-HCl, and 5 mg liter�1 lipoic acid. All vitamins wereadded from sterile-filtered stock solutions. For the isotope studies, wereplaced glucose with an equimolar amount of 99% [1-13C]glucose (Eu-risotop, Saarbrücken, Germany). Oxidative stress studies were conductedusing soft-agar assays (59). Minimal medium containing 15 g liter�1 agar(Becton Dickinson) was covered with a 1:1 mixture of soft agar (7.5 gliter�1 agar in a 2.4% [wt/vol] NaCl solution) and minimal medium withresuspended cells. Filter discs (5-mm diameter) soaked with 10 �l of di-amide [1,1-azo-bis(N,N-dimethylformamide)] (0.6 M in dimethyl sul-foxide [DMSO]) were then placed onto the homogeneous layer of cells.

Cultures. Cultures with [1-13C]glucose were prepared in a volumeof 2 ml using deep-well plates (riplate BV [10 ml]; HJ-Bioanalytik,Mönchengladbach, Germany) and incubated at 1,000 rpm on a plateshaker (Inkubator 1000; Heidolph Instruments, Schwabach, Germany).To avoid evaporation, the plates were sealed with gas-permeable adhesivemembranes (HJ-Bioanalytik). Single colonies from agar plates that wereincubated for 72 h were used to inoculate the first preculture. After incu-bation for 12 h, the cells were harvested by centrifugation (15,700 � g, 5min, 4°C), washed with sterile 0.9% (wt/vol) NaCl, and used as the inoc-ulum for the second preculture. The main cultures were inoculated usingexponentially growing cells from the second preculture that were washedas described above. For each strain, 12 parallel cultures were prepared.Samples from four wells were pooled, yielding three biological replicatesof each strain for subsequent analysis. The growth performance was vali-dated with respect to the metabolic steady state (see Fig. S1 and Fig. S2 inthe supplemental material). In selected cases, the main cultures were ad-ditionally grown in three biological replicates (1-liter baffled shake flasks,100 ml of minimal medium) and incubated on a rotary shaker (230 rpm).These cultures were used to provide sufficient amounts of cells for gravi-metric analysis of the cell weight (dry weight) (cdw) or enzymatic mea-surements. Oxidative stress tests were performed in triplicate. The agarplates with diamide were incubated in the dark. All cultures were grown at30°C.

Quantification of cell concentrations. The cell concentrations weremonitored based on the optical density at 600 nm (OD600). For P. inhi-bens, the cdw was additionally measured after the collection of cells bycentrifugation (9,000 � g, 10 min, 4°C); the cells were washed with 0.9%(wt/vol) NaCl, and the pellet was dried at 80°C until it reached a constantweight. Both measurements were integrated to determine the correlationbetween the optical density and cell weight (dry weight, in grams per liter)(equation 3).

cdw � OD600 � 0.2657 (3)

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This correlation was used to estimate the culture volume required toobtain 10 to 30 mg of dry biomass based on the measured optical density.This amount was appropriate for the subsequent 13C gas chromatography(GC)-mass spectrometry (MS) analysis. The correlation determined for P.inhibens was applied to all strains.

Enzyme assays. The cells were harvested during the exponentialgrowth phase (9,000 � g, 10 min, 4°C), washed with disruption buffer(200 mM Tris-HCl [pH 8.0], 200 mM KCl, 50 mM K3PO4-NaOH, 0.75mM dithiothreitol [DTT], 1 mM ATP, and 5 mM MgCl2 in 5% [vol/vol]glycerol) and disrupted mechanically (twice for 4 min each time, 30 Hz)(Ribolyzer MM301; Retsch, Haan, Germany). The cell debris was re-moved by centrifugation (16,000 � g, 5 min, 4°C). The protein concen-tration in the obtained extract was determined using a bicinchoninic acid(BCA) assay kit (Thermo Fisher Scientific, Bonn, Germany). The phos-phofructokinase activity (EMP pathway) was quantified in a reaction mixcontaining 100 mM Tris-HCl (pH 8.0), 50 mM K3PO4-NaOH, 32.5 mMKCl, 5 mM MgCl2, 0.25 mM NADH, 0.1 mM ATP, 0.5 U of aldolase, 0.5U of glyceraldehyde 3-phosphate dehydrogenase, 1 U of triose phosphateisomerase, 4 mM fructose 6-phosphate, and 0.02 mg ml�1 protein. Thereaction mix for quantification of glucose 6-phosphate dehydrogenaseactivity (joint reaction of PP pathway and ED pathway) contained 100mM Tris-HCl (pH 7.8), 200 mM KCl, 1 mM NAD(P), 10 mM MgCl2, 5mM glucose 6-phosphate (G6P), and 50 �l cell extract in a total volume of1 ml (42). The enzyme activity was determined by monitoring the forma-tion of NAD(P)H at 340 nm. The reaction mix for the determination of6-phosphogluconate dehydrogenase (PP pathway) activity contained 100mM Tris-HCl buffer (pH 7.8), 10 mM MgCl2, 0.75 mM DTT, 1 mMEDTA, 2.5 mM 6-phosphogluconate, 1 mM NAD(P)H, and 0.04 mg ml�1

protein. The level of NAD(P)H was monitored by measuring the absor-bance at 340 nm (Sunrise; Tecan Group, Switzerland), and the change inabsorbance was used to calculate the enzymatic activity. Negative controlslacked substrate and cell extract.

Mass spectrometric labeling analysis by GC-MS. Cells were collectedby centrifugation (9,000 � g, 10 min, 4°C) and washed with deionizedwater. Prior to GC-MS analysis, the cellular protein was hydrolyzed for 24h at 105°C using 50 �l of 6 M HCl per mg of cells (dry weight). Cell debriswas removed by filtration (Ultrafree-MC; Millipore, Billerica, MA, USA).The labeling patterns of proteinogenic amino acids were analyzed usingtheir t-butyldimethylsilyl (TBDMS) derivatives (43) by GC-MS (Agilent7890A and quadrupole mass selective detector 5975C; Agilent Technolo-gies, Waldbronn, Germany). The samples were first measured in the scan-ning mode to check for isobaric overlay in the ion clusters of interest,which might interfere with the labeling of the amino acids. Subsequently,selective ion monitoring was used for quantitative analysis. Three individ-ual runs, each with a different subset of measured ion clusters and repre-senting technical duplicates, were conducted per sample (see Fig. S3 in thesupplemental material). The amino acid mass distributions were obtainedfrom the spectra after correction for the natural abundance of stable iso-topes (44). For each strain, the data set contained the relative fractions of84 different mass isotopomers (see Table S2 in the supplemental mate-rial).

Multivariate data analysis. The amino acid labeling patterns cor-rected for natural isotope abundances were subjected to independentcomponent analysis (ICA) (45). In the analysis of isotope experimentdata, ICA automatically recognizes conserved and biologically relevantlabeling patterns, as demonstrated in a previous analysis of Bacillus subtilismutants (46). ICA assumes that the labeling patterns result from the su-perposition of independent metabolic activities; therefore, each activitycauses a shift in the mass distribution of one or more metabolites. ICAseparates the observed variables into non-Gaussian statistically indepen-dent components (ICs), which allows for the identification of the masssignals of metabolites that enable discrimination based on metabolic dif-ferences. We used the publicly available FastICA 2.1 algorithm (HUT-CIS; http://research.ics.aalto.fi/ica/fastica/) in Matlab (R2010b; Math-works, Natick, MA, USA) to derive the ICs. Subsequently, ICASSO

bootstrapping was applied to validate the reliability and robustness of theidentified ICs (47, 48).

Calculation of the relative fluxes of the glucose catabolism path-ways. The relative contributions of the EMP, ED, and PP pathways toglucose catabolism were assessed from the 13C labeling pattern of pro-teinogenic alanine, generated during growth on [1-13C]glucose (24). Theanalysis considered the relative fraction of the nonlabeled mass isotopom-ers (M0) of the entire alanine molecule with carbon atoms C1, C2, and C3

(Ala123) and of a fragment that contained the two carbon atoms C2 and C3

(Ala23). These were obtained from mass spectrometric analysis ofTBDMS-derivatized alanine at a mass-to-charge (m/z) ratio for themonoisotopic mass of 260 (Ala123) and 232 (Ala23) (see Table S2 in thesupplemental material). After correction for natural isotope abundances,the relative catabolic pathway fluxes into the ED pathway (fEDP), the EMPpathway (fEMPP), and the PP pathway (fPPP) were derived using the fol-lowing algebraic equations.

fPPP � 2(M0,Ala123� 0.5) (4)

fEMPP � �2(M0,Ala23� 1) (5)

fEDP � 1 � fPPP � fEMPP (6)

If the calculation yielded negative values, results were corrected tozero. The calculations assumed that the reactions were not reversible. Thecalculation of the respective fluxes via the labeling of serine and alanine(24) yielded the same results (data not shown).

Phylogenetic, functional cluster, and statistical analyses. Clusteranalysis of amino acid mass distributions was conducted using Matlab(R2010b; Statistical Toolbox, Mathworks) to derive a Euclidean distancetree for the different strains based on their metabolic properties. In addi-tion, cluster analysis was performed using the ARB software package (49)(www.arb-home.de) based on 16S rRNA sequences to derive a compara-tive phylogenetic tree. The sequences of the type strains (�1,300 bp) wereused to construct the backbone tree using the maximum likelihoodmethod. Subsequently, shorter sequences from the isolates were added byinteractive parsimony. The studied isolates were then integrated into thephylogenetic tree. Differences between experimental data were statisti-cally evaluated for significance by a t test (Origin 9.1; OriginLab, North-ampton, MA, USA).

RESULTSStrain selection, labeling strategy, and isotope experiments forresolution of glycolytic pathway flux. For our study, we selected25 marine isolates of Alphaproteobacteria, Gammaproteobacteria,and Flavobacteriia of the Bacteriodetes phylum and Actinobacteriaas representative classes of marine bacterial communities, includ-ing strains that were recently sequenced (Fig. 1; see Table S1 in thesupplemental material). The isolates (Fig. 1) originated from dif-ferent geographical marine regions such as the North Sea, Medi-terranean Sea, East China Sea, and Sea of Japan, and covered dif-ferent ecological niches, including open water, sediment, andintertidal mudflats, coastal regions, and the surface of macroalgae.All strains were able to utilize glucose as the sole carbon source(see Fig. S1 and Fig. S2 in the supplemental material). For com-parison, well-known terrestrial bacteria that use glucose (E. coli, C.glutamicum, P. putida, B. subtilis, and B. megaterium) were ana-lyzed. Briefly, the relative fluxes through the alternative pathwaysof glucose catabolism in the strains of interest were assessed frommass spectrometric analysis of alanine from the cellular protein ofcells, grown on [1-13C]glucose as the tracer substrate (Fig. 2). Eachof the three catabolic pathways results in a different 13C labelingpattern of pyruvate, the precursor of alanine: the ED pathwayresults in a mixture of nonlabeled pyruvate and [3-13C]pyruvate,the EMP pathway results in a mixture of nonlabeled pyruvate and

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[1-13C]pyruvate, and the PP pathway results in nonlabeled pyru-vate. Consequently, a unique combination of labeling patterns inalanine fragments, which contain carbons C1C2C3 (Ala123) andC2C3 (Ala23), respectively, is observed for each pathway (Fig. 2).This forms the basis of the algebraic equations 4 to 6, which linkthis particular labeling information with relative pathway flux(24). The two fragments Ala123 and Ala23 are accessible as ionclusters at m/z 260 and m/z 232 of TBDMS-derivatized alanine,respectively. To elucidate catabolic glucose fluxes, the strains werenow cultivated in defined seawater medium with [1-13C]glucoseas a tracer substrate. The analysis of glucose consumption and cellgrowth revealed that the biological replicates for each strain grew

highly reproducibly (Table S3). Furthermore, the specific growthrate and the biomass yield coefficient for each strain were constantover time and reflected metabolic steady state (Fig. S1 and Fig. S2).Obviously, all batch cultures fulfilled the requirement to recruitthe labeling of amino acids from cell protein to derive valid andconsistent fluxes (50).

Marine microorganisms predominantly use the ED pathwayas the glycolytic route. For each strain, comprehensive labelingdata sets were collected (see Table S2 in the supplemental mate-rial). Qualitative inspection of the labeling data has already re-vealed that the marine strains differed in glucose catabolism. Forexample, Pseudonocardiaceae bacterium T4 possessed 13C-en-

FIG 1 Phylogenetic tree showing the relationships of the marine bacteria studied based on 16S rRNA gene sequence similarity. The sequences of type strains(�1,300 bp) were used to construct the backbone tree using maximum likelihood. Subsequently, shorter sequences from the isolates studied were added byinteractive parsimony. GenBank sequence accession numbers are given in parentheses. The strains investigated in this study are shown in bold type (see also TableS4 in the supplemental material).

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riched alanine fragments Ala23 and Ser123, which unambiguouslyindicated an active EMP pathway in this particular strain, whereasthe same fragments were not 13C enriched but naturally labeled inother marine isolates. The theoretical framework of the underly-ing carbon transfer through the biochemical reactions involved(equations 4 to 6) and the 13C labeling of the two specific alaninefragments (Table S2) provided the relative fluxes through theEMP, ED, and PP pathways on a quantitative basis. The analysisrevealed an interesting picture (Fig. 3). Sixteen strains near exclu-sively used the ED pathway, whereas the EMP and PP pathwayswere inactive (marine cluster I in Fig. 3). These strains belonged to

all studied marine clades and families, i.e., Gammaproteobacteria(Pseudoalteromonas marina, Pseudoalteromonas haloplanktis,Pseudoalteromonas nigrifaciens HEL-36, Pseudoalteromonas sp.HEL-40, Pseudoalteromonas sp. HEL-49, Alteromonas macleodii,Alteromonas distincta HEL-05, Alteromonas macleodii HEL-51, Al-teromonas sp. BIO-267, Alteromonas sp. BIO-296, and Gamma-proteobacteriaceae bacterium sp. GWS-SE-H233), Alphaproteo-bacteria (Phaeobacter inhibens and Dinoroseobacter shibae), andFlavobacteriia (Polaribacter dokdonensis and Leeuwenhoekiella sp.Pic90). P. putida was the only terrestrial strain that showed thisflux pattern. Another subgroup of seven marine isolates used the

FIG 2 Labeling strategy for discrimination of relative carbon fluxes through the Entner-Doudoroff (ED) pathway, Embden-Meyerhof-Parnas (EMP) pathway,and pentose phosphate (PP) pathway from an isotope experiment with [1-13C]glucose as the tracer substrate. Different carbon transitions lead to a differentlabeling pattern of pyruvate for each pathway and result in unique labeling patterns of the two alanine fragments [M-57] and [M-85], which contain the carbonatoms C1C2C3 and C2C3 of pyruvate, respectively. This information can be used to infer flux information via the algebraic equations given in Materials andMethods. Further details are given elsewhere (24). 6P-Gluconate; 6-phosphogluconate; Glucose 6-P, glucose 6-phosphate.

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ED pathway and the oxidative part of the PP pathway in parallelbut did not exhibit any EMP flux (marine cluster II). This fluxprofile also occurred among all clades: Gammaproteobacteria(Pseudomonas sp. GWS-TZ-H209 and Oceanospirillaceae bacte-rium T17), Alphaproteobacteria (Jannaschia sp. B3, Loktanella sp.D3, and Phyllobacteriaceae bacterium TK), and Flavobacteriia(Flavobacteriaceae bacterium TN and Flavobacteriaceae bacteriumT15). A third cluster exhibited predominant use of the EMP routeas the glycolytic pathway for glucose metabolism; the PP pathwayoperated in parallel with the EMP pathway, albeit to a lower ex-tent, and the ED pathway was completely inactive. Notably, thisflux pattern was observed for the majority of the terrestrial strains,including E. coli, C. glutamicum, and the two bacilli, and only threeorganisms of marine origin (Eudoraea adriatica, Vibrio sp. GWS-TZ-H304, and Pseudonocardiaceae bacterium T4). The individualstrains assigned to marine cluster III differed to some extent re-garding the relative contribution of the EMP and PP pathways;therefore, members of this cluster grouped not as tightly as mem-bers of the other two clusters. Overall, the results indicated a pref-erence of marine bacteria to use the ED pathway as the sole ormajor catabolic route. In contrast, most of the studied terrestrialmicrobes used the EMP pathway.

In P. putida, D. shibae, and P. inhibens, the absence of phos-phofructokinase prevents the use of the EMP pathway for glucosecatabolism (24, 51), whereas E. coli, C. glutamicum, B. subtilis, andB. megaterium possess this enzyme and exhibit a functional EMPpathway (17, 52–54). We now examined whether the observeddifferences in pathway usage among the marine isolates also re-lates to their enzyme inventory or to pathway regulation and thuschecked for in vitro activity of phosphofructokinase. Almost allisolates that preferred or exclusively used the ED pathway indeedlacked phosphofructokinase activity (Table 1), whereas the en-zyme was expressed in the strains with a functional EMP pathway.The entry enzyme of the PP pathway, 6-phosphogluconate dehy-drogenase, was expressed in all active PP pathway users, but noenzyme activity was observed in almost all strains that did not usethis pathway (Table 1). Taken together, the absence of functionalEMP and PP pathways seemed to be due to a lack of phosphofruc-tokinase and 6-phosphogluconate dehydrogenase expression, re-spectively, rather than due to control of these enzymes on themetabolic level. In the latter case, one would have expected a mea-surable in vitro activity. This excludes the possibility that theseenzymes are expressed but metabolically controlled. This did,however, not necessarily correlate with the absence of the corre-

FIG 3 Relative fluxes through the major pathways of glucose catabolism in marine and nonmarine bacterial species. Relative fluxes through the major pathwaysof glucose catabolism, the Embden-Meyerhof-Parnas (EMP) pathway, Entner-Doudoroff (ED) pathway, and pentose phosphate (PP) pathway, in 25 marinebacteria (blue symbols) and 8 nonmarine bacteria studied in this work (green symbols) or previous work (yellow symbols) are shown. Marine cluster I bacteriaexclusively use the ED pathway. Marine cluster I bacteria include Phaeobacter inhibens, Dinoroseobacter shibae, Alteromonas macleodii, Polaribacter dokdonensis,Pseudoalteromonas haloplanktis, Pseudoalteromonas marina, Pseudoalteromonas sp. HEL-36, Pseudoalteromonas sp. HEL-40, Pseudoalteromonas sp. HEL-49,Leeuwenhoekiella sp. Pic90, Alteromonas sp. HEL-5, Alteromonas sp. HEL-51, Alteromonas sp. BIO-267, Alteromonas sp. BIO-296, and Gammaproteobacteriaceaebacterium GWS-SE-H233. Nonmarine P. putida and P. aeruginosa PAO1 also use the ED pathway (16). Marine cluster II bacteria use the ED and PP pathwaysin parallel and include Flavobacteriaceae bacterium TN, Phyllobacteriaceae bacterium TK, Flavobacteriaceae bacterium T15, Oceanospirillaceae bacterium T17,Jannaschia sp. B3, Loktanella sp. D3, and Pseudomonas sp. GWS-TZ-H209. Marine cluster III bacteria use the EMP and PP pathways in parallel and includeEudoraea adriatica, Vibrio sp. GWS-TZ-H304, and Pseudonocardiaceae bacterium T4. The parallel use of the EMP and PP pathways is also found for nonmarineBacillus subtilis (this work) (19), Bacillus megaterium (this work), Escherichia coli (this work) (90), Corynebacterium glutamicum (this work) (52), Yersiniapseudotuberculosis (91), and Sorangium cellulosum (92).

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sponding gene, as deduced from genome sequence information(Table 2). Pathway use and enzymatic set matched with the ge-netic repertoire for only three of the sequenced isolates, i.e., Dino-roseobacter shibae, P. inhibens, and Eudoraea adriatica. In contrast,P. haloplanktis, P. marina, and A. macleodii did not reveal 6-phos-phogluconate dehydrogenase activity, though corresponding genes

are annotated. In addition, Alteromonas macleodii did not exhibit6-phosphofructokinase activity, but it has an annotated gene.

Most marine strains possess glucose 6-phosphate dehydro-genase with flexible cofactor use. For the marine strains, glucose6-phosphate dehydrogenase appeared to be a key enzyme for cat-abolic glucose breakdown, because alternative routes were not

TABLE 1 Specific activities of phosphofructokinase, glucose 6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase in marinebacteria

Strain or species Phylogenetic class Family Clustera

Sp act (mU mg�1)b

PFK

G6PDH

GNDNADP NAD

Pseudoalteromonas sp. HEL-36 Gammaproteobacteria Pseudoalteromonadaceae I �0.1 906 � 49 286 � 47 �0.1Pseudoalteromonas sp. HEL-40 Gammaproteobacteria Pseudoalteromonadaceae I �0.1 516 � 11 81 � 9 �0.1Pseudoalteromonas sp. HEL-49 Gammaproteobacteria Pseudoalteromonadaceae I �0.1 488 � 20 97 � 8 �0.1Alteromonas sp. HEL-5 Gammaproteobacteria Alteromonadaceae I �0.1 616 � 81 181 � 6 �0.1Alteromonas sp. HEL-51 Gammaproteobacteria Alteromonadaceae I �0.1 322 � 24 52 � 15 �0.1Alteromonas sp. BIO-267 Gammaproteobacteria Alteromonadaceae I �0.1 681 � 46 256 � 11 �0.1Alteromonas sp. BIO-296 Gammaproteobacteria Alteromonadaceae I �0.1 665 � 23 256 � 7 �0.1Gammaproteobacterium H233 Gammaproteobacteria Halomonadaceaee I �0.1 307 � 24 61 � 8 �0.1Phaeobacter inhibens Alphaproteobacteria Rhodobacteraceae I �0.1 50 � 4 14 � 8 �0.1Dinoroseobacter shibae Alphaproteobacteria Rhodobacteraceae I �0.1 101 � 4 33 � 4 �0.1Leeuwenhoekiella sp. Pic90 Flavobacteriia Flavobacteriaceae I �0.1 319 � 13 �0.1 �0.1Pseudoalteromonas haloplanktis Gammaproteobacteria Pseudoalteromonadaceae I �0.1 374 � 43 60 � 4 �0.1Pseudoalteromonas marina Gammaproteobacteria Pseudoalteromonadaceae I �0.1 498 � 54 40 � 12 �0.1Alteromonas macleodii Gammaproteobacteria Alteromonadacea I �0.1 183 � 35 62 � 9 �0.1Oceanospirillaceae bacterium T17 Gammaproteobacteria Oceanospirillaceae II �0.1 33 � 6 �0.1 2 � 0Pseudomonas sp. GWS-TZ-H209 Gammaproteobacteria Pseudomonadaceae II �0.1 342 � 45 �0.1 19 � 1Jannaschia sp. B3 Alphaproteobacteria Rhodobacteraceae II �0.1 240 � 22 45 � 7 95 � 20Loktanella sp. D3 Alphaproteobacteria Rhodobacteraceae II �0.1 1,287 � 7 705 � 7 16 � 1Phyllobacteriaceae bacterium TK Alphaproteobacteria Phyllobacteriaceae II �0.1 366 � 14 247 � 37 29 � 1Pseudonocardiaceae bacterium T4 Actinobacteria Pseudonocardiaceae III 117 � 9 �0.1 �0.1 20 � 1Vibrio sp. GWS-TZ-H304 Gammaproteobacteria Vibrionaceae III 155 � 31 299 � 79 �0.1 61 � 5Eudoraea adriatica Flavobacteriia Flavobacteriaceae III 128 � 4 63 � 20 �0.1 182 � 16a The analyzed strains or species were assigned to cluster I (use of the ED pathway only), cluster II (parallel use of the ED and PP pathways), or cluster III (parallel use of the EMPand PP pathways) according to their catabolic flux profiles (Fig. 3).b Specific activity of phosphofructokinase (PFK) (in the EMP pathway), glucose 6-phosphate dehydrogenase (G6PDH) (in a joint reaction of ED and PP pathway), and 6-phosphogluconate dehydrogenase (GND) (in the PP pathway) in marine bacteria. The data are means � standard deviations from three biological replicates. Cytosolic extractsfrom Polaribacter doktonensis and Flavobacteriaceae bacterium TN and T15 did not contain sufficient protein for reliable measurement; therefore, these strains were excluded fromthe enzymatic analysis. The enzyme assays were conducted under saturated in vitro conditions.

TABLE 2 Pathway repertoire of sequenced members among the investigated marine bacteriaa

Species Genome identifier

Locus tag(s)b

PFK G6PDH GND PntAB

Phaeobacter inhibens PhaInh 188638 NA 0943, 3373 NA 1476, 1477Dinoroseobacter shibae DinShi 9476 NA 1748 NA 1233, 1234Pseudoalteromonas haloplanktis PsaHal NA 3806 0916 NAPseudoalteromonas marina PseMar 220774 NA 2870 2687 NAAlteromonas macleodii AltMac 49397 2216 1233 1244 3857–3859Eudoraea adriatica EudAdr 278662 0029, 0030 0875 0876 1916–1918Polaribacter dokdonensisc PolSp 11901 0658 NA 1940 NAa Pathway repertoire of sequenced members among the investigated marine bacteria, extracted through the bacterial bioinformatics database and analysis resource PATRIC (55).The data given comprise genome identifier and locus tags of genes annotated as 6-phosphofructokinase (PFK), glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconatedehydrogenase (GND), and NADPH-forming membrane-bound transhydrogenase PntAB, respectively. For PFK and G6PDH, duplicate entries reflect putative isoenzymes. ForPntAB, the given tags represent different open reading frames for the individual subunits of the enzyme complex. Here, a potentially functional enzyme was attributed only tostrains, which comprised both subunits A and B.b NA, not annotated.c The genome sequence was obtained from the closely related isolate Polaribacter sp. MED-152, a marine bacterium that was isolated from the surface water of northwesternMediterranean Sea off the Catalan coast (56). In the original GenBank submission, it was listed as a strain of Polaribacter dokdonensis (31), with which it has 99.6% similar 16SrRNA sequence.

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expressed or even not encoded. All ED pathway users, i.e., 90% ofthe studied marine isolates, channeled their substrate carbon en-tirely via this biochemical reaction. Likewise, the strains that ex-hibited a split use of the EMP pathway and the PP pathway obvi-ously needed the enzyme to direct carbon into the PP pathway. Invitro analysis of glucose 6-phosphate dehydrogenase revealed thatthe enzyme was expressed in all marine strains studied. Loktanellasp. D3 had the highest activity (1.3 U mg�1), and for most otherstrains, the enzyme activity was in the range of 0.3 to 0.6 U mg�1.A few isolates accepted only NADP as a cofactor for glucose6-phosphate dehydrogenase. These included the EMP users Eudo-raea adriatica, Vibrio sp. GWS-TZ-H304, and selected strainsfrom clusters I and II (Table 1). It was interesting to note thatglucose 6-phosphate dehydrogenase showed activity with NADPand NAD in most marine strains, whereby NADP was the pre-ferred cofactor. The NAD-related activity of the enzyme in thesestrains was variable and ranged from 8% (P. marina) to 67% (Lok-tanella sp. D3) of the NADP-related activity. In addition, the PPpathway enzyme 6-phosphogluconate dehydrogenase was studiedfor cofactor prevalence. All strains of clusters II and III, i.e., withan active PP pathway (Fig. 2), were analyzed accordingly. It turnedout that 6-phosphogluconate dehydrogenase accepted onlyNADP as a cofactor but was not active with NAD (data notshown).

Marine Gammaproteobacteria differ from marine Alphapro-teobacteria and Flavobacteria bacteria in the anaplerotic fluxesthat replenish the TCA cycle. In bacteria, the phosphoenolpyru-vate (PEP)-pyruvate-oxaloacetate node is a central switch pointfor the distribution of carbon flux among the catabolic, anabolic,and energy supply pathways (57). Bacteria differ in the types ofreactions occurring at this node, that is, certain bacteria can re-plenish the tricarboxylic acid (TCA) cycle via the carboxylation ofeither PEP, pyruvate, or both. The following labeling strategy al-lowed elucidation of the type of anaplerotic metabolism in 90% ofthe marine strains, i.e., in all strains that used the ED pathway. As

recently demonstrated, PEP- and pyruvate-based anaplerotic me-tabolism provides a unique combination of 13C enrichment inconnected pathway intermediates of such bacteria using the EDpathway grown on [1-13C]glucose (16). Considering the well-de-fined transition of carbon atoms in the biochemical reactions in-volved, high 13C enrichment in pyruvate, together with low 13Cenrichment in oxaloacetate results in PEP carboxylation, whereasthe opposite results in pyruvate carboxylation, respectively (Fig.4). The labeling pattern of alanine, reflecting its precursor pyru-vate, and of aspartate, reflecting its precursor oxaloacetate, re-vealed two distinct groups: marine Gammaproteobacteria obvi-ously use PEP carboxylase, whereas Alphaproteobacteria andFlavobacteria recruit pyruvate carboxylase.

Metabolic pathway use coincides with oxidative stress toler-ance. Many habitats in the marine environment impart oxida-tive stress; therefore, antioxidant mechanisms are importanttraits of marine microorganisms (58). To study the tolerance ofmarine bacteria to such oxidative conditions, we studied thetolerance to diamide (59). Diamide causes oxidative stress byoxidizing sulfhydryl bonds in the cytoplasm, which must bereduced at the expense of NADPH (60, 61). Here, we used aplate-based assay with diamide applied to a filter disc in thecenter of the plate. The area of the resulting halo of growthinhibition provided a direct measure of the sensitivity of thecells to oxidative stress (Fig. 5A). P. putida has robust resistanceto oxidative stress (59). Thus, the tolerance of P. putida was used asthe reference (100%) to classify the other tested bacteria accordingto their stress tolerance. Notably, most marine strains had a hightolerance to oxidative stress (Fig. 5A, middle and lower rows, andB). Certain isolates, e.g., Alteromonas sp. BIO-267, even exhibiteda higher tolerance than P. putida. In comparison, E. adriatica,Vibrio sp. GWS-TZ-H304, and Pseudonocardiae bacterium T4 hadsubstantially weaker tolerance and had a sensitivity equal to that ofE. coli (Fig. 5A, top row, and B). Statistical analysis revealed thatthe ED pathway users in particular had a high stress tolerance

FIG 4 Anaplerotic fluxes in all strains studied that use the ED pathway for glucose catabolism. (A) The activity of PEP carboxylase causes a low 13C enrichmentof oxaloacetate (aspartate), and 13C preferentially accumulates in pyruvate (alanine). G6P, glucose 6-phosphate; 6-PG, 6-phosphogluconate; KDPG, 2-keto-3-deoxy-6-phosphogluconate; GAP, glycerol-3-phosphate; PYR, pyruvate; OAA, oxaloacetate. (B) However, the activity of pyruvate carboxylase causes lower 13Cenrichment in pyruvate (alanine) and higher enrichment in oxaloacetate (aspartate). Further details are described in reference 16. (C) The strains cluster into twogroups based on the labeling patterns of aspartate and alanine, both shown as the relative fraction of the single-labeled (M1) mass isotopomers. The strains thatutilize pyruvate carboxylase are shown in orange, whereas the strains that use PEP carboxylase are shown in green.

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(Fig. 5B, blue bars), whereas the strains that preferred the EMProute (Fig. 5B, red bars) were significantly more sensitive to oxi-dative stress (P � 0.05 by t test).

Cluster analysis of 13C labeling signatures provides a meta-bolic degree of similarity among the isolates. The aforemen-tioned analysis of proteinogenic 13C labeling data using algebraicequations provided direct insight into selected flux properties:glucose catabolism and anaplerotic metabolism. Subsequently, weexpanded our view of central metabolism by considering the en-tire set of 13C amino acid labeling data from the isotope experi-ments (see Fig. S4 in the supplemental material). Using P. inhibensas a reference, we observed that more than 90% of the marinestrains had similar patterns with less than 20% variation in indi-vidual mass isotopomer fractions. The 13C fingerprints of thesestrains were different from those of the three marine strains Eu-doraea adriatica, Vibrio sp. GWS-TZ-H304, and Pseudonocardi-aceae bacterium T4 and from those of E. coli, C. glutamicum, andthe two bacilli. A systematic and unsupervised analysis of the la-beling data was conducted using a statistical approach. We per-formed an independent component analysis (ICA) of the data set.This analysis identified 24 specific signatures in the labeling pat-terns, that is, the independent components (ICs), which were in-formative in describing and differentiating the metabolism of thestrains analyzed (see Fig. S5 and Fig. S6 in the supplemental ma-terial). On the basis of this finding, we then used a cluster analysisof the entire amino acid labeling data set (Table S2) to quantify thedegree of similarity between the marine isolates. The resultingdendrogram (Fig. 6) shows that the degree of similarity on thelevel of 13C labeling matched with metabolic function; the identi-

fied metabolic strategies among the studied isolates clustered to-gether nicely.

DISCUSSION

Because marine microbes have a capacity for rapid growth, theyare a major component of global nutrient cycles. In their naturalhabitats, marine bacteria often have to face fluctuating conditions,scarce nutrient levels, and oxidative stress due to absorption ofsolar radiation (25, 62, 63). Questions regarding how their distri-bution is controlled and the diverse repertoire of nutrient trans-formations are major challenges, faced by contemporary biologi-cal oceanographers (64). In particular, glucose assimilation plays amajor role, as marine bacteria may represent up to 40% of bacte-rial carbon production in the oceans (5). Here, we present a met-abolic flux approach to study the carbon metabolism in marinebacteria that use glucose. The 25 selected strains represented glob-ally distributed marine clades and families (26, 65, 66) such asAlteromonadaceae, Pseudoalteromonadaceae (Gammaproteobacte-ria), Rhodobacteraceae (Alphaproteobacteria), Flavobacteria, andSphingobacteria of the Bacteriodetes phylum and Actinobacteria(Fig. 1 and Table 1).

Marine bacteria prefer the ED pathway as a glycolytic strat-egy. A central finding of our study is the strong prevalence of theED pathway among the marine bacteria investigated. More than90% of them rely on the ED pathway as the sole or major glycolyticroute (Fig. 3) and even lack a functional EMP pathway due to theabsence of phosphofructokinase activity, although this does notnecessarily imply the absence of the gene itself (Table 1). In con-trast, the EMP pathway is nearly ubiquitous in the bacterial king-

FIG 5 Evaluation of oxidative stress tolerance by treatment with diamide. (A) Soft-agar plates with a central diamide-containing filter disc were photographedafter 24 h of incubation. (B) To obtain a quantitative measure of stress resistance, the area of the resulting circular cell-free zone of inhibition or halo wascalculated from the measured diameters of three replicates. The data were normalized to the halo area of P. putida (100%). The Flavobacterium isolate T15 didnot form a homogeneous cell layer; therefore, representative data for this species could not be obtained.

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dom (13), and recent genomic analyses of more than 500 micro-bial species revealed that only 12% of prokaryotes rely solely onthe ED pathway (14). In line with this, metabolic flux studies usingvarious bacteria, including aerobic C. glutamicum (52) and B. sub-tilis (67), the facultative anaerobes E. coli (68) and Basfia succi-niciproducens (69), and the anaerobes Lactobacillus plantarum(70) and Lactococcus lactis (71), identified the EMP pathway as themajor catabolic pathway during growth on glucose. It has beenproposed that the ED pathway may not play a major role in glu-cose metabolism but instead primarily functions in the break-down of sugar acids that cannot be metabolized through the EMPpathway (14, 72). However, this conclusion seems not applicableto marine bacteria (this work). Marine microbes, at least on thebasis of the broad collection of strains from different clades stud-ied, instead form a specific subgroup among the prokaryotes withregard to catabolic pathway use. This observation is striking be-cause the EMP pathway yields twice as much ATP as the ED path-way does and therefore appears superior (Fig. 7A). Interestingly,other phylogenetically distinct bacteria (Alphaproteobacteria andGammaproteobacteria) also rely on the ED pathway for glucosecatabolism (Fig. 3) (15, 16). The obvious use of the ED pathway bysuch a diverse group of bacteria suggests that this type of metab-olism has a greater importance in nature, as was previously recog-nized (72), and might confer other advantages for cellular metab-

olism that overcome the drawback of a lower ATP yield. It isinteresting to note that glucose 6-phosphate dehydrogenase activ-ity was not specific for NADP in the ED pathway users (Table 1).Generally, this can be explained by glucose 6-phosphate dehydro-genases without preference for either cofactor or by differentisoenzymes with different cofactor specificities (73). On the basisof the available genomic data, it becomes clear that most strainshave a promiscuous glucose 6-phosphate dehydrogenase, becausethey possess a unique gene encoding it, with P. inhibens being theonly exception (Table 2). This property may relate to the fact thatthe enzyme is required for the exclusive glucose catabolic pathway inthese species, and therefore, its nonspecificity serves as a major mech-anism to avoid catabolic NADPH overproduction (73). It should benoticed that, due to experimental requirements, the glucose level inthe flux studies was higher than that naturally found in seawater (4,5). Those species that possess the genetic repertoire for different gly-colytic pathways (Table 2) might be able to switch pathway use, whenglucose is scarce. However, the majority of the marine isolates thatlack phosphofructokinase (Tables 1 and 2) seem to be bound to theED pathway, independent of the nutrient level.

PEP and pyruvate carboxylation are evenly distributedamong marine bacteria that use glucose. The type of anapleroticstrategy has been shown to influence the flexibility under chang-ing conditions (57). This seems to be of particular importance for

FIG 6 Cluster analysis of marine isolates based on the amino acid labeling fingerprints. The corresponding phylum for each strain is indicated by the coloredtriangle as follows: dark purple for Gammaproteobacteria, light purple for Alphaproteobacteria, light blue for Flavobacteriia, and black for Actinobacteria. Inaddition, the metabolic strategies identified are shown. These strategies include the glycolytic route (EMPP [red] and EDP [blue]), the anaplerotic route (PEPcarboxylase [green] and pyruvate carboxylase [orange]), and the use of the oxidative PP pathway (PPP) (active [turquoise] and not active [pink]). For strainsusing the EMP pathway, the anaplerotic pathway could not be elucidated (shown in white).

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life in the highly heterogeneous oceans, which exhibit constantlychanging physical and chemical gradients (25). In contrast to aclear preference for the catabolic ED route, the marine bacteriastudied did not specifically rely on a distinct anaplerotic route, butthe carboxylation of PEP or pyruvate for fueling of the TCA cyclewas evenly distributed among the strains. We conclude that bothstrategies provide sufficient flexibility for bacteria that use glucoseto adapt to the changing conditions in the sea.

The ED pathway provides redox power to protect against ox-idative stress. Oxidative stress is common in the oceans and iscaused by the absorption of solar radiation (62) and the me-tabolism of marine algae (74). Marine bacteria possess variousantioxidant mechanisms to eliminate reactive oxygen species(ROS), the inducers of oxidative stress (58). Particularly,NADPH is an oxidative stress protectant that is required bymany important antioxidant defense mechanisms (75) and isimportant for counteracting oxidative stress (61, 76, 77). Arecent study revealed the importance of the ED pathway foroxidative stress protection in P. putida (59). As impressivelyshown, the introduction of a functional phosphofructokinaseforced the organism to shift the flux from the natural ED pathwayto the EMP pathway, which significantly decreased its toleranceto oxidative stress. Similarly, the human pathogen Pseudomo-nas aeruginosa exclusively uses the ED pathway and potentiallyprovides far more NADPH than needed for anabolism: a benefit tocounteract oxidative stress imposed by the host during infection

(16). Based on these findings, it is tempting to speculate that theconserved use of the ED pathway in marine bacteria might simi-larly support their high tolerance to oxidative stress through ele-vated NADPH formation. Notably, all ED pathway users possess asuperior tolerance to oxidative stress, whereas strains that utilizethe EMP pathway have a weaker stress tolerance (Fig. 5). Althoughthe lack of high-level NADPH specificity for the glucose 6-phos-phate dehydrogenase (Table 1) does not exclude at least a partialformation of NADH with the ED pathway in the correspondingstrains, the nonspecificity of the enzyme surely enables the organ-ism to cope with dynamic fluctuations in NADP and NADPHavailability (73, 75). Especially under conditions of oxidativestress, cells exhibit a drastically disturbed redox equilibrium andthe NADPH/NADP ratio is reduced almost 10-fold (75). Onecan expect that this strongly promotes NADPH formation by glu-cose 6-phosphate dehydrogenase. Assuming stoichiometric for-mation of NADPH by glucose 6-phosphate dehydrogenase, whichappears reasonable for such stress conditions, the resulting stoi-chiometry for the ED and EMP pathways, the quantified flux par-titioning ratio (Fig. 2), and the identified enzymatic inventory(Table 1) allow for the estimation of the supply of reducing power(NADPH) and energy (ATP) under conditions of oxidative stress.With the given assumptions, all ED pathway users would supplylarge amounts of NADPH (Fig. 7), which could explain their highrobustness (Fig. 5), whereas strains that utilize the EMP pathwayand generate less NADPH (Fig. 7) have a weaker stress tolerance

FIG 7 Generation of NADPH and ATP in the strains studied, calculated based on the measured metabolic fluxes (Fig. 2) and the stoichiometry of theEmbden-Meyerhof-Parnas (EMP), Entner-Doudoroff (ED), and pentose phosphate (PP) pathways. (A) The reactions are catalyzed by glucose 6-phosphateisomerase (step 1), phosphoglucokinase (step 2), fructose 1,6-bisphosphate aldolase (step 3), glucose 6-phosphate dehydrogenase (step 4), phosphogluconatedehydratase (step 5), 2-keto-3-desoxy-6-phosphogluconate aldolase (step 6), lumped reactions of GAP dehydrogenase, phosphoglycerate kinase, phosphoglyc-erate mutase, and enolase (step 7), and pyruvate kinase (step 8). F6P, fructose 6-phosphate; FBP, fructose 1,6-bisphosphate. (B) The calculation of NAPDHgeneration was based on the formation of one and two NADPH molecules via the ED and PP pathways, respectively. The estimation of ATP production was basedon the direct formation of 3 ATP (EMP), 2 ATP (EDP), and 8/3 ATP (PPP) and indirect formation via the oxidation of NADH formed in the respiratory chainat a P/O ratio of 2 (93). The stoichiometry for NADH production is 2 NADH (EMP), 1 NADH (EDP), and 5/3 NADH (PPP). Prior to the estimation, themeasured fluxes were corrected on the basis of the enzymatic repertoire (Table 1), i.e., small EMP fluxes predicted by the algebraic calculation were set to zerowhen phosphofructokinase was absent, and this flux was then attributed to the ED pathway.

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(Fig. 5). Particularly, members of the Roseobacter clade, Altero-monadaceae, Pseudoalteromonadaceae, and Flavobacteriaceae, allED pathway users (Fig. 3), were found to be highly tolerant tooxidative conditions (Fig. 5) (78) and are abundant and active inmarine surface waters that receive high light intensities and im-pose severe oxidative stress (79, 80). Similarly, members of theRoseobacter clade (81), Flavobacteriaceae (82), and Alteromon-adaceae and Pseudoalteromonadaceae (83) live physically attachedto marine algae, which are also strong inducers of oxidative stressrelated to the release of ROS during photosynthesis (74, 84).Clearly, bacterial cells possess a variety of mechanisms for manag-ing oxidative stress (85), and one cannot fully discern the contri-butions of ED pathway-derived reducing power derived fromother mechanisms within the cell, such as glutathione and variousenzymes. Owing to the central importance of NADPH as thedriver of many of these mechanisms, it is, however, likely that lifein oxidative marine environments at least partially benefits fromthe enhanced NADPH supplied via the ED pathway and is onemajor reason why this pathway is dominating in the marine bac-teria studied.

A significant contribution of transhydrogenase PntAB, whichmight potentially contribute to NAPDH formation, seems un-likely. Careful inspection of the genomic data does not support acentral role of this enzyme. For example, bacteria with high ro-bustness, i.e., P. haloplanktis, P. marina, and P. dokdonensis, do notcomprise pntAB genes in their genome, whereas stress-sensitivebacteria such as E. adriatica and E. coli are equipped with pntABgenes (Table 2).

The EMP pathway might be beneficial in anoxic marine en-vironments. Among the marine strains, Pseudonocardiaceae T4and Vibrio sp. GWS-TZ-H304 used the EMP pathway (Fig. 3).Notably, both bacteria were isolated from low-oxygen habitats.Pseudonocardiaceae T4 grows in an intertidal mudflat (35) knownto have low oxygen levels below the surface (86). Vibrio sp. GWS-TZ-H304 was originally isolated from an oxic-anoxic sedimentboundary (37), and other Gammaproteobacteria have been foundin oxygen-minimum zones (87, 88). In these habitats, oxidativestress and nonglycolytic energy generation are low, and energyefficiency rather than redox efficiency determines the glycolyticstrategy; therefore, a functional EMP pathway might be beneficial(14).

Marine bacteria cluster according to their metabolic flux pat-terns. As shown, the 13C signatures contained discriminatory in-formation that can distinguish between the strains on a metabolicbasis (Fig. 6). The dendrogram derived indicated two majorgroups, which differ in their catabolic pathway use: the EMP andED pathway users. The latter group separates into two subgroupswith regard to anaplerotic metabolism. In this regard, it is inter-esting to note that the metabolic similarity determined does notalways correlate with the phylogenetic similarity (Fig. 6). We sug-gest taking such a metabolic viewpoint in order to obtain morefunctional insight into the role or similarity of the cells examined,complementary to that based on DNA sequences. This would re-flect the observation that the physiology of microbes, of whichmetabolism is a key component, might be the key driver for theirevolution (89). Regarded from this viewpoint, the Alteromonasand Pseudoalteromonas strains of Gammaproteobacteria clusteredwith Leeuwenhoekiella sp. Pic90, a Flavobacterium; all of thesestrains used PEP carboxylase. Other Gammaproteobacteria, i.e.,Pseudomonas sp. GWS-TZ-H209 and Oceanospirillaceae bacte-

rium T17, used pyruvate carboxylase, as did most of the Alphapro-teobacteria. As recently suggested for environmental bacteria,their main evolutionary drive is the expansion of their metabolicnetworks toward new chemical landscapes rather than perpetua-tion and spreading of their DNA sequences (89).

Conclusions. Unlike terrestrial ecosystems, microorganismsare the main form of biomass in the oceans and comprise thelargest living surface on the planet (26). Most of the microbes thatuse glucose that were studied rely on the ED pathway to survive inmarine environments. Due to the fact that glucose is a major nu-trient in seawater (5), the ED pathway may play a significant rolein global biogeochemical cycles (13, 14).

ACKNOWLEDGMENTS

This work was funded by Deutsche Forschungsgemeinschaft within theframework of the Collaborative Research Centre “Roseobacter” (TRR51).

We declare that we have no conflicts of interest.A.K. conducted growth and 13C labeling experiments. A.K. and J.B.

conducted metabolic flux analysis and statistical analysis of 13C labelingpatterns. A.B. and A.K. performed enzymatic assays. A.K., A.B., J.B., andC.W. performed oxidative stress experiments. M.D., M.S., and T.B. per-formed the phylogenetic analysis. M.S., T.B, M.D., and I.W.-D. providedmarine isolates. A.K., D.J., M.S., I.W.-D., T.B., J.B., and C.W. interpretedthe data, drafted the manuscript, and critically revised it for importantintellectual content. C.W. designed and supervised the study. We all readand approved the final manuscript.

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