Data_Sheet_1_Chemiosmotic Energy Conservation in Dinoroseobacter shibae: Proton Translocation Driven by Aerobic Respiration, Denitrification, and Photosynthetic Light Reaction.DOCX

<p>Dinoroseobacter shibae is an aerobic anoxygenic phototroph and able to utilize light energy to support its aerobic energy metabolism. Since the cells can also grow anaerobically with nitrate and nitrite as terminal electron acceptor, we were interested in how the cells profit from photosynthesis during denitrification and what the steps of chemiosmotic energy conservation are. Therefore, we conducted proton translocation experiments and compared O₂^-, NO₃^-, and NO₂^- respiration during different light regimes and in the dark. We used wild type cells and transposon mutants with knocked-out nitrate- and nitrite- reductase genes (napA and nirS), as well as a mutant (ppsR) impaired in bacteriochlorophyll a synthesis. Light had a positive impact on proton translocation, independent of the type of terminal electron acceptor present. In the absence of an electron acceptor, however, light did not stimulate proton translocation. The light-driven add-on to proton translocation was about 1.4 H⁺/e^- for O₂ respiration and about 1.1 H⁺/e^- for NO₃^- and NO₂^-. We could see that the chemiosmotic energy conservation during aerobic respiration involved proton translocation, mediated by the NADH dehydrogenase, the cytochrome bc₁ complex, and the cytochrome c oxidase. During denitrification the last proton translocation step of the electron transport was missing, resulting in a lower H⁺/e^- ratio during anoxia. Furthermore, we studied the type of light-harvesting and found that the cells were able to channel light from the green–blue spectrum most efficiently, while red light has only minor impact. This fits well with the depth profiles for D. shibae abundance in the ocean and the penetration depth of light with different wavelengths into the water column.</p

Robustness and Plasticity of Metabolic Pathway Flux among Uropathogenic Isolates of <i>Pseudomonas aeruginosa</i>

<div><p><i>Pseudomonas aeruginosa</i> is a human pathogen that frequently causes urinary tract and catheter-associated urinary tract infections. Here, using ¹³C-metabolic flux analysis, we conducted quantitative analysis of metabolic fluxes in the model strain <i>P. aeruginosa</i> PAO1 and 17 clinical isolates. All <i>P. aeruginosa</i> strains catabolized glucose through the Entner-Doudoroff pathway with fully respiratory metabolism and no overflow. Together with other NADPH supplying reactions, this high-flux pathway provided by far more NADPH than needed for anabolism: a benefit for the pathogen to counteract oxidative stress imposed by the host. <i>P. aeruginosa</i> recruited the pentose phosphate pathway exclusively for biosynthesis. In contrast to glycolytic metabolism, which was conserved among all isolates, the flux through pyruvate metabolism, the tricarboxylic acid cycle, and the glyoxylate shunt was highly variable, likely caused by adaptive processes in individual strains during infection. This aspect of metabolism was niche-specific with respect to the corresponding flux because strains isolated from the urinary tract clustered separately from those originating from catheter-associated infections. Interestingly, most glucose-grown strains exhibited significant flux through the glyoxylate shunt. Projection into the theoretical flux space, which was computed using elementary flux-mode analysis, indicated that <i>P. aeruginosa</i> metabolism is optimized for efficient growth and exhibits significant potential for increasing NADPH supply to drive oxidative stress response.</p></div

Kinetics and stoichiometry of glucose-grown <i>P. aeruginosa</i> PAO1 and uropathogenic <i>P. aeruginosa</i> isolates obtained from patients with catheter-associated urinary tract infection or urinary tract infections.

<p>The corresponding cultivation profiles for all strains are provided in Supporting Information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088368#pone.0088368.s003" target="_blank">Figure S3</a>). Values indicate means and standard deviations of three biological replicates. By-products in the supernatants were not detected; concentrations were below the detection limit (1 µM for amino acids and 10 µM for organic acids).</p

Metabolic adaption of pathogenic <i>P. aeruginosa</i>.

<p>The network representation integrates changes of flux determined for uropathogenic isolates and <i>P. aeruginosa</i> PAO1 (this work) and changes in transcription transcript level previously determined for successive isolates of <i>P. aeruginosa</i> from patients with chronic cystic fibrosis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088368#pone.0088368-Hoboth1" target="_blank">[11]</a>. The variation of metabolic flux among strains is indicated by color (black = conserved, green = changed). Genes with changed or unchanged transcript levels are shown in green or black, respectively. Abbreviations are as follows: Entner-Doudoroff pathway (EDP), Embden-Meyerhof-Parnas pathway (EMPP), pentose phosphate pathway (PPP), glyoxylate (Gly) shunt, and tricarboxylic acid (TCA) cycle.</p

Metabolic properties of uropathogenic <i>P. aeruginosa</i> isolates and <i>P. aeruginosa</i> PAO1.

<p>Integration of flux phenotypes into the theoretical flux space on basis of anabolism (considering the biomass yield coefficient Y_x/s, given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088368#pone-0088368-t001" target="_blank">Table 1</a>) and of NADPH metabolism (considering the NADPH balance as described below). In order to evaluate individual flux phenotypes, experimental values for biomass yield and apparent NADPH excess, respectively, (black squares) are integrated into the flux space created by elementary flux mode analysis (grey squares). Most strains localize close to the growth optimum. (A), Correlation of biomass formation and NADPH metabolism (B), contribution of isocitrate dehydrogenase and 6-phosphogluconate dehydrogenase to supply of reducing power (C), correlation of biomass formation and flux through the oxidative pentose phosphate pathway (D). NADPH metabolism was inspected by balancing of the redox cofactor. For this purpose, the NADPH formation flux by concerted action of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase, and malic enzyme was balanced with the NADPH consumption flux for anabolism. For all strains, NADPH formation was higher than consumption. The resulting apparent excess flux of NADPH supply is presented here. The full flux data sets for all strains are presented in Supporting Information. The flux values are normalized to the corresponding glucose uptake rate for each strain (set to 100%).</p

Metabolic flux in <i>P. aeruginosa</i> PAO1 and select gram-negative and gram-positive bacteria.

<p>Flux refers to the Entner-Doudoroff pathway (EDP), Embden-Meyerhof-Parnas pathway (EMPP), pentose phosphate pathway (PPP), and glyoxylate (Gly) shunt and reflects relative values normalized to the corresponding glucose uptake rate, defined as 100%.</p><p>* functional pathway not encoded.</p><p>** n.d. = not determined.</p

Niche-specific metabolic flux in uropathogenic <i>P. aeruginosa</i>, including flux through isocitrate dehydrogenase and isocitrate lyase (A) and through pyruvate kinase and phosphoenolpyruvate carboxykinase (B).

<p>Each data point refers to a distinct isolate. The clustering represents statistical analysis using principal components (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088368#pone-0088368-g004" target="_blank">Figure 4</a>) that discriminated between isolates from catheter-associated infections (cluster <i>a</i>) and strains from urinary tract infections (clusters <i>b</i> and <i>c</i>). The full flux data sets for all strains are presented in Supporting Information. All flux values are normalized to the average glucose uptake rate for all strains (100%).</p

Statistical analysis of carbon core metabolism of uropathogenic <i>P. aeruginosa</i> isolates and <i>P. aeruginosa</i> PAO1 based on ¹³C-labeled amino acid enrichment data from the tracer studies.

<p>Principal component analysis provided a clustering of the strains according to the two major components (A). Hierarchical cluster analysis revealed the degree of similarity, shown as a Euclidian tree (B). The relative fraction of the single labeled (M1) mass isotopomer of each amino acid was considered after normalization to the value of <i>P. aeruginosa</i> PAO1. The relative ¹³C-enrichment is displayed in color. The amino acids are denoted by their single letter code.</p

In vivo carbon flux distributions in central metabolism of uropathogenic <i>P. aeruginosa</i> isolates during growth on glucose.

<p>Flux is given as average flux of all strains and is expressed as a molar percentage of the average glucose uptake rate of all strains (8.6 mmol g⁻¹ h⁻¹, calculated from the individual rates in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088368#pone-0088368-t001" target="_blank">Table 1</a>). Open arrows indicate flux toward biomass. For reversible reactions, the direction of net flux is indicated by a dashed arrow. The errors given for each flux reflect the corresponding 90% confidence intervals. The full flux data sets are presented in Supporting Information. Metabolic and isotopic steady states are ensured by constant stoichiometry, kinetics, and the constant ¹³C-labeling patterns of recruited metabolites during cultivation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088368#pone.0088368.s001" target="_blank">Figure S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088368#pone.0088368.s005" target="_blank">Figure S5</a>). Abbreviations are as follows: Entner-Doudoroff pathway (EDP), Embden-Meyerhof-Parnas pathway (EMPP), pentose phosphate pathway (PPP), glyoxylate (Gly) shunt, and tricarboxylic acid (TCA) cycle.</p

In vivo carbon fluxes through central metabolic pathways of uropathogenic <i>P. aeruginosa</i> during growth on glucose.

<p>The data reflect the individual flux for each isolate. All fluxes are expressed as a molar percentage of the corresponding specific glucose uptake rate (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088368#pone-0088368-t001" target="_blank">Table 1</a>). Statistical differences of flux between strains from urinary tract infections and from catheter-associated infections were assessed using a <i>t</i> test. Significant differences (**<i>P</i><0.05) are indicated. Abbreviations are as follows: glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), 6-phosphogluconate dehydratase (EDD), isocitrate dehydrogenase (ICDH), isocitrate lyase (ICL), pyruvate kinase (PK), pyruvate dehydrogenase (PDH), malic enzyme (ME), pyruvate carboxylase (PC), and phosphoenolpyruvate carboxykinase (PEPCK). The full flux data sets for all strains are presented in the Supporting Information.</p