Role of central metabolism in the osmoadaptation of the halophilic bacterium Chromohalobacter salexigens

Abstract

Bacterial osmoadaptation involves the cytoplasmic accumulation of compatible solutes to counteract extracellular osmolarity. The halophilic and highly halotolerant bacterium Chromohalobacter salexigens is able to grow up to 3 m NaCl in a minimal medium due to the de novo synthesis of ectoines. This is an osmoregulated pathway that burdens central metabolic routes by quantitatively drawing off TCA cycle intermediaries. Consequently, metabolism in C. salexigens has adapted to support this biosynthetic route. Metabolism of C. salexigens is more efficient at high salinity than at low salinity, as reflected by lower glucose consumption, lower metabolite overflow, and higher biomass yield. At low salinity, by-products (mainly gluconate, pyruvate, and acetate) accumulate extracellularly. Using [1-(13)C]-, [2-(13)C]-, [6-(13)C]-, and [U-(13)C6]glucose as carbon sources, we were able to determine the main central metabolic pathways involved in ectoines biosynthesis from glucose. C. salexigens uses the Entner-Doudoroff pathway rather than the standard glycolytic pathway for glucose catabolism, and anaplerotic activity is high to replenish the TCA cycle with the intermediaries withdrawn for ectoines biosynthesis. Metabolic flux ratios at low and high salinity were similar, revealing a certain metabolic rigidity, probably due to its specialization to support high biosynthetic fluxes and partially explaining why metabolic yields are so highly affected by salinity. This work represents an important contribution to the elucidation of specific metabolic adaptations in compatible solute-accumulating halophilic bacteria.

Keywords: Ectoines; Entner-Doudoroff; Halophilic Bacteria; Metabolic Tracers; Metabolism; Microbiology; Nuclear Magnetic Resonance; Overflow Metabolism; Pyruvate Carboxylase; Salt Adaptation.

FIGURE 1.

Formation of biomass (black circles) and extracellular concentrations of glucose (black squares), gluconate (white circles), pyruvate (white squares), and acetate (white triangles) of batch cultures grown in 20 mm glucose minimal medium with 0.6 m NaCl (A), 0.75 m NaCl (B), and 2.5 m NaCl (C).

FIGURE 2.

Effect of NaCl concentration on production of ectoines and consumption of carbon and nitrogen sources in C. salexigens. A, cellular contents of ectoine and hydroxyectoine. B, relative content of proteins and ectoines, expressed as percentage of total pool of ectoines plus proteins. The sum of these pools was approximately constant throughout all conditions tested (0.423 ± 0.055 g/g_CDW). C, stoichiometric coefficient of glucose (dark bars) and ammonium consumption (light bars). Molar ratio of ammonium to glucose utilization is denoted by circles. Cultures were grown at 37 °C in M63 minimal medium with 20 mm glucose and 30 mm ammonium as the sole carbon and nitrogen sources, respectively. See the text for details.

FIGURE 3.

Scheme of the central metabolism and synthesis of ectoines in C. salexigens based on the annotated genome. Pathways leading from glucose to 6-P-gluconate (6PGln) are proposed on the comparison of the in silico analysis of C. salexigens and P. putida. Abbreviations used are as follows: 2-KGlcn, 2-ketogluconate; 2-Kglu, 2-ketoglutarate; 6PGlcn, 6-phospho-d-gluconate; 6PKGlcn, 6-phospho-2-keto-d-gluconate; AcCoA, acetyl-coenzyme A; AcP, acetyl phosphate; Ala, l-alanine; Amm, ammonium; Asp, l-aspartate; Asp-P, l-aspartyl phosphate; G3P, glyceraldehyde 3-phosphate; Glc, d- glucose; Glc6P, d-glucose 6-phosphate; Glcn, d-gluconate; Glcnlac, d-gluconolactone; Gln, l-glutamine; Gox, glyoxylate; Glu, l-glutamate; Ict, d-isocitrate; KDGlcn6P, 2-keto-3-deoxy-d-gluconate-6-phosphate; Lac, d-lactate; Lys, l- lysine; Mal, l-malate; Met, l-methionine; NADA, N-γ-acetyl-l-2,4-diaminobutyrate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; Pyr, pyruvate; Suc, succinate; Thr, l-threonine; Acs, acetyl-coenzyme A synthetase; ActP, acetate permease; AcyP, acetyl phosphate phosphatase; AlaAT, alanine aminotransferase; Ald, alanine dehydrogenase; AspAT, aspartate aminotransferase; AspK, aspartate kinase; Cs, citrate synthase; EctA, diaminobutyrate acetyltransferase; Gad, gluconate dehydrogenase; Gdhq, glucose dehydrogenase; Gdh, glutamate dehydrogenase; Glk, glucokinase; Gls, glutamate synthase; GnuK, gluconokinase; Icl, isocitrate lyase; KguD, 2-keto-6-phosphogluconate reductase; KguK, 2-ketogluconate kinase; Ldh, lactate dehydrogenase; Mae, malic enzyme; Mqo, malate-quinone oxidorreductase; Ms, malate synthase; Oad, oxaloacetate decarboxylase; Pc, pyruvate carboxylase; Pdh, pyruvate dehydrogenase; Pox, pyruvate oxidase; Ppc, phosphoenolpyruvate carboxylase; Pyk, pyruvate kinase; Zwf, glucose-6-phosphate dehydrogenase. Dashed arrows are used for conversions that require more than one enzymatic step.

FIGURE 4.

¹³C NMR spectra of intracellular extracts of [1-¹³C]glucose-grown cultures. M63 minimal medium with 0.75 m NaCl (upper spectrum) and 2.5 m NaCl (lower spectrum) was used. The signals corresponding to labeled carboxylic carbon (177 ppm for ectoine and 174–175 for hydroxyectoine) and C6 (38–39 ppm for ectoine and 44 for hydroxyectoine) are shown. In the scheme, the expected fate of labeled carbon when [1-¹³C]glucose is metabolized via the Entner-Doudoroff pathway is shown. If we assume that the labeled C1 from pyruvate is lost as ¹³CO₂ by decarboxylation at the level of Pdh and incorporated into OAA by Pc, the predicted ectoines labeling pattern would fit the spectra obtained. Relative labeling for each carbon atom is indicated by the color scale at right. The abbreviations used are as follows: labeled compounds detected: G, glutamate; E, ectoine; H, hydroxyectoine; GLC, glucose; PYR, pyruvate; OAA, oxaloacetate; AcCoA, acetyl-coenzyme A.

FIGURE 5.

Incorporation of label from [2-¹³C]glucose into ectoines. ¹³C from [2-¹³C]pyruvate (derived via either the Embden-Meyerhof or the Entner-Doudoroff pathways), from [2-¹³C]phosphoenolpyruvate (made via Embden-Meyerhof), or from unlabeled phosphoenolpyruvate (made via Entner-Doudoroff) can be incorporated into ectoines through the following. A, oxaloacetate synthesized in a single TCA cycle turn; B, oxaloacetate synthesized by pyruvate carboxylase or phosphoenolpyruvate carboxylase (Pc/Ppc), or (C) oxaloacetate synthesized by Pc/Ppc followed by a turn through the TCA cycle, which alters its labeling pattern (see the text for details). D, scheme depicting the relation of the pyruvate and acetyl-CoA nodes with the ectoines biosynthesis route in C. salexigens. E, ¹³C NMR spectra from intracellular extracts of [2-¹³C]glucose-grown cultures. M63 minimal medium with 0.75 m NaCl (upper spectrum) and 2.5 m NaCl (lower spectrum) was used. The signals corresponding to labeled carboxyl carbon, methyl carbon, C2, C4, and C6 of ectoine (E) and hydroxyectoine (H) are shown. The signal corresponding to hydroxyectoine carboxyl carbon overlaps with that of C1 of glutamate (indicated as C1, G). Note that for each pair of chemical shifts corresponding to each carbon, the ratio of the ectoine/hydroxyectoine signals is approximately constant. The three ectoine molecules in the inset represent the isotopomer distributions corresponding to a–c. Relative labeling for each carbon atom is indicated by the color scale at right of E. Where applicable, the upper half of the corresponding carbon position ball depicts the expected labeling from [2-¹³C]pyruvate/[2-¹³C]phosphoenolpyruvate, and the lower half from unlabeled phosphoenolpyruvate. See supplemental Table S4 and Fig. S5 and supplemental material “Determination of Metabolic Flux Ratios” for details.