A Functional Tricarboxylic Acid Cycle Operates during Growth of Bordetella pertussis on Amino Acid Mixtures as Sole Carbon Substrates

Abstract

It has been claimed that citrate synthase, aconitase and isocitrate dehydrogenase activities are non-functional in Bordetella pertussis and that this might explain why this bacterium's growth is sometimes associated with accumulation of polyhydroxybutyrate (PHB) and/or free fatty acids. However, the sequenced genome includes the entire citric acid pathway genes. Furthermore, these genes were expressed and the corresponding enzyme activities detected at high levels for the pathway when grown on a defined medium imitating the amino acid content of complex media often used for growth of this pathogenic microorganism. In addition, no significant PHB or fatty acids could be detected. Analysis of the carbon balance and stoichiometric flux analysis based on specific rates of amino acid consumption, and estimated biomass requirements coherent with the observed growth rate, clearly indicate that a fully functional tricarboxylic acid cycle operates in contrast to previous reports.

Fig 1. Kinetics of growth of B. pertussis Tohama I in batch cultures.

Results are displayed as mean values for the three biological replicates with a regular Y-scale (a) and a logarithmic Y-scale (b). Error bars represent the replicates' standard deviations. Dry cell weight concentrations have been calculated from OD measurements at 650 nm.

Fig 2. Kinetics of amino acid consumption of B. pertussis Tohama I in batches cultures.

(a): Kinetics of glutamate and proline; (b) kinetics of aromatic amino acids; (c): kinetics of amino acids derived from pyruvate; (d): kinetics of amino acids derived from oxaloacetate. Results are displayed as mean values for the three biological replicates and error bars represent standard deviations.

Fig 3. Estimation of carbon distribution within the central metabolic pathways based on the kinetic data from three biological replicates of B. pertussis Tohama I batch cultures.

(a) Growth period: 0–12h; (b) Growth period: 12h-end of culture. G6P: glucose-6-phosphate; F6P: fructose-6-phosphate; R5P: ribulose-5-phosphate; E4P: erythrose-4-phosphate; GAP: glyceraldehyde-3-phosphate; 3PG: 3-phosphoglycerate; PEP: phosphoenolpyruvate; PYR: pyruvate; AcCoA: acetyl-CoA; CIT: citrate, ISOC: isocitrate; αKG: α-ketoglutarate; SUC: succinate; MAL: malate; OAA: oxaloacetate; GLU: glutamate; PRO: proline; AA: amino acids; Phe: phenylalanine, Tyr: tyrosine; Ala: alanine; Gly: glycine; Ile: isoleucine; Leu: leucine; Ser: serine; Val: valine; Asp: aspartate; Lys: lysine; Met: methionine; Thr: threonine. Numerical data are reported for the three replicates as mean ± standard deviation. They are expressed in mmoles per liter and per gram of biomass (mM/gX).