Environmental and nutritional factors that affect growth and metabolism of the pneumococcal serotype 2 strain D39 and its nonencapsulated derivative strain R6

Abstract

Links between carbohydrate metabolism and virulence in Streptococcus pneumoniae have been recurrently established. To investigate these links further we developed a chemically defined medium (CDM) and standardized growth conditions that allowed for high growth yields of the related pneumococcal strains D39 and R6. The utilization of the defined medium enabled the evaluation of different environmental and nutritional factors on growth and fermentation patterns under controlled conditions of pH, temperature and gas atmosphere. The same growth conditions impacted differently on the nonencapsulated R6, and its encapsulated progenitor D39. A semi-aerobic atmosphere and a raised concentration of uracil, a fundamental component of the D39 capsule, improved considerably D39 growth rate and biomass. In contrast, in strain R6, the growth rate was enhanced by strictly anaerobic conditions and uracil had no effect on biomass. In the presence of oxygen, the difference in the growth rates was mainly attributed to a lower activity of pyruvate oxidase in strain D39. Our data indicate an intricate connection between capsule production in strain D39 and uracil availability. In this study, we have also successfully applied the in vivo NMR technique to study sugar metabolism in S. pneumoniae R6. Glucose consumption, end-products formation and evolution of intracellular metabolite pools were monitored online by (13)C-NMR. Additionally, the pools of NTP and inorganic phosphate were followed by (31)P-NMR after a pulse of glucose. These results represent the first metabolic profiling data obtained non-invasively for S. pneumoniae, and pave the way to a better understanding of regulation of central metabolism.

Figure 1. Growth profiles of strains D39 and R6 in chemically defined medium with and without pH control.

Growth of strains D39 (□) and R6 (▪) in CDM containing 60 mM glucose, without pH control (initial pH of 6.5), at 37°C, under semi-aerobic conditions, in static rubber-stoppered bottles (V, 80 ml) (A) or under controlled conditions of pH (6.5), temperature (37°C) and atmosphere (B) semi-aerobiosis (C) anaerobiosis (D) aerobiosis, in a 2-l bioreactor. The arrows in (A) indicate the time-points at which cells were harvested for measurement of NADH oxidase and LDH activities. The growth rate for each culture is also indicated and the values are averages ± SD.

Figure 2. Schematic overview of the pathways for glucose metabolism and capsule production in S. pneumoniae.

Glucose is oxidized to pyruvate via the Embden-Meyerhof-Parnas pathway (glycolysis); a common metabolic intermediate in glycolysis is glucose 6-phosphate (G6P), which is also a precursor for the biosynthesis of capsule NDP-sugars; NDP-sugars are synthesized at the expense of UTP. Pyruvate is the substrate of three competing enzymes: lactate dehydrogenase, pyruvate formate-lyase and pyruvate oxidase. Homolactic fermentation reduces pyruvate into lactate, through lactate dehydrogenase (LDH), whereas mixed-acid fermentation leads to formate, acetate and ethanol. Oxygen might be consumed at the level of lactate oxidase (LOX coded by lctO), pyruvate oxidase (SpxB) or H₂O-NADH oxidase (NOX). The occurrence of the pyruvate dehydrogenase complex (PDHc) depicted in grey remains to be proved. Proposed pathways were reconstructed based on genome database (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi), literature and database surveys (KEGG, MetaCyc). Gene annotation downloaded from NCBI: nox, NADH oxidase; pyk, pyruvate kinase; ldh, L-lactate dehydrogenase; lctO, lactate oxidase; spxB, pyruvate oxidase; ackA, acetate kinase; pfl, pyruvate formate-lyase; pta, phosphotransacetylase; adh, bifunctional acetaldehyde-CoA/alcohol dehydrogenase; PDHc, putative pyruvate dehydrogenase complex.

Figure 3. Fermentation profiles of strains D39 and R6 under aerobic conditions.

Growth curves, substrate consumption and end-products formed by the D39 (A) and R6 (B) strains growing aerobically as in Fig. 2C. Culture supernatant samples for end-product analysis by HPLC and/or ¹H-NMR were harvested during growth. Symbols: (○), Glucose consumption; (▪), growth curve; (♦), lactate; (▴), acetate; (•), H₂O₂. The error was below 7% for major products (>2 mM) and 25% for minor products (<2 mM).

Figure 4. Growth profiles of D39 and R6 at different glucose concentrations.

Growth of strains D39 (A) and R6 (B) in CDM containing (♦) 0.5%, (▪) 1% or (•) 3% (wt/vol) glucose, under controlled conditions of pH (6.5), temperature (37°C) and atmosphere (semi-aerobiosis), in a 2-l bioreactor. The growth rate for each culture is also indicated and the values are averages ± SD.

Figure 5. Effect of nucleobases concentration on growth profiles.

Growth of strains D39 (A) and R6 (B) in CDM containing 1% (wt/vol) glucose, under controlled conditions of pH (6.5), temperature (37°C), and atmosphere (semi-aerobiosis), in a 2-l bioreactor. The nucleobases were added to the medium as follows: (▪), G, A, X, U 10 mg l⁻¹ each; (▴), G, A, X, U 30 mg l⁻¹ each; (•), G, A, X 10 mg l⁻¹ each plus 30 mg l⁻¹ U. G = guanine; A = Adenine; X = Xanthine; U = Uracil.

Figure 6. Effect of varying uracil concentration on growth of strain D39.

(A) Growth of strain D39 in CDM containing 1% (wt/vol) glucose with different concentrations of uracil (U), as specified below. Growth was performed at 37°C, without pH control (initial pH 6.5), under semi-aerobic conditions. Uracil concentrations in mg l⁻¹: (□), 0; (▵), 0.67; (⋄), 1; (○), 3.3; (▴), 5; (▪), 10; (•), 30; (♦), 40. The other three nucleobases (G, X, A) were always present in the medium at a concentration of 10 mg l⁻¹. G = guanine; X, xanthine; A, adenine. (B) Linear correlation between the maximal D39 biomass (OD_Max) and the medium uracil concentration (from 0.67 to 10 mg l⁻¹).

Figure 7. Glucose metabolism in resting cell suspensions of S. pneumoniae R6 under anaerobic conditions monitored by in vivo NMR.

(A) Kinetics of 20 mM [1-¹³C]glucose consumption, end-products formation and build-up of glycolytic intermediate pools by R6 resting cells as monitored in vivo by ¹³C-NMR (B) Time course for the concentration of NTP (▴) and total inorganic phosphate, P_i (•) monitored in vivo by ³¹P-NMR during glucose (20 mM) metabolism of R6 resting cells. Symbols: (♦), glucose; (□), lactic acid; (○), acetate; (⋄), glycerol; (▵), fructose 1,6-bisphosphate.