Carbon source-dependent reprogramming of anaerobic metabolism in Staphylococcus aureus

To be a successful pathogen, S. aureus has to adapt its metabolism to the typically oxygen- and glucose-limited environment of the host. Under fermenting conditions and in the presence of glucose, S. aureus uses glycolysis to generate ATP via substrate level phosphorylation and mainly lactic acid fermentation to maintain the redox balance by re-oxidation of NADH equivalents. However, it is less clear how S. aureus proceeds under anoxic conditions and glucose limitation, likely representing the bona-fide situation in the host. Using a combination of proteomic, transcriptional and metabolomic analyses, we show that in the absence of an abundant glycolysis substrate the available carbon source pyruvate is converted to acetyl-CoA (AcCoA) in a pyruvate formate-lyase (PflB)-dependent reaction to produce ATP and acetate. This process critically depends on de-repression of the catabolite control protein A (CcpA), leading to upregulation of pflB transcription. Under these conditions, ethanol production is repressed to prevent wasteful consumption of AcCoA. In addition, our global and quantitative characterization of the metabolic switch prioritizing acetate over lactate fermentation when glucose is absent illustrates examples of carbon source-dependent control of colonization and pathogenicity factors. Importance: Under infection conditions, S. aureus needs to ensure survival when energy production via oxidative phosphorylation is not possible, e.g. either due to the lack of terminal electron acceptors or by the inactivation of components of the respiratory chain. Under these conditions, S. aureus can switch to mixed acid fermentation to sustain ATP production by substrate-level phosphorylation. The drop in the cellular NAD+/NADH ratio is sensed by the repressor Rex, resulting in de-repression of fermentation genes. Here we show that expression of fermentation pathways is further controlled by CcpA in response to the availability of glucose to ensure optimal resource utilization under growth limiting conditions. We provide evidence for carbon source-dependent control of colonization and virulence factors. These findings add another level to the regulatory network controlling mixed acid fermentation in S. aureus and provide additional evidence for the lifestyle-modulating effect of carbon sources available in S. aureus.

On-line analysis and in situ pH monitoring of mixed acid fermentation by Escherichia coli using combined FTIR and Raman techniques

We introduce an experimental setup allowing continuous monitoring of bacterial fermentation processes by simultaneous optical density (OD) measurements, long-path FTIR headspace monitoring of CO2, acetaldehyde and ethanol, and liquid Raman spectroscopy of acetate, formate, and phosphate anions, without sampling. We discuss which spectral features are best suited for detection, and how to obtain partial pressures and concentrations by integrations and least squares fitting of spectral features. Noise equivalent detection limits are about 2.6 mM for acetate and 3.6 mM for formate at 5 min integration time, improving to 0.75 mM for acetate and 1.0 mM for formate at 1 h integration. The analytical range extends to at least 1 M with a standard deviation of percentage error of about 8%. The measurement of the anions of the phosphate buffer allows the spectroscopic, in situ determination of the pH of the bacterial suspension via a modified Henderson-Hasselbalch equation in the 6–8 pH range with an accuracy better than 0.1. The 4 m White cell FTIR measurements provide noise equivalent detection limits of 0.21 μbar for acetaldehyde and 0.26 μbar for ethanol in the gas phase, corresponding to 3.2 μM acetaldehyde and 22 μM ethanol in solution, using Henry’s law. The analytical dynamic range exceeds 1 mbar ethanol corresponding to 85 mM in solution. As an application example, the mixed acid fermentation of Escherichia coli is studied. The production of CO2, ethanol, acetaldehyde, acids such as formate and acetate, and the changes in pH are discussed in the context of the mixed acid fermentation pathways. Formate decomposition into CO2 and H2 is found to be governed by a zeroth-order kinetic rate law, showing that adding exogenous formate to a bioreactor with E. coli is expected to have no beneficial effect on the rate of formate decomposition and biohydrogen production.

Altered Superoxide Dismutase Activity by Carbohydrate Utilization in a Lactococcus lactis Strain

Reactive oxygen species, such as superoxide, can damage cellular components, such as proteins, lipids, and DNA. Superoxide dismutase (SOD) enzymes catalyze the conversion of superoxide anions to hydrogen peroxide and dioxygen. SOD is present in most lactococcal bacteria, which are commonly used as starters for manufacturing fermented dairy products and may have health benefits when taken orally. We assessed the effects of carbohydrate use on SOD activity in lactococci. In Lactococcus lactis ssp. lactis G50, the SOD activity of cells grown on lactose and galactose was higher than that on glucose; in Lactococcus lactis ssp. cremoris H61, SOD activity was independent of the type of carbohydrate used. We also investigated the activity of NADH oxidase, which is related to the production of superoxide in strains G50 and H61. Activity was highest in G50 cells grown on lactose, lower on galactose, and lowest on glucose, whereas activity in H61 cells did not differ with the carbohydrate source used. The SOD and NADH oxidase activities of strain G50 in three carbohydrates were linked. Strain G50 fermented lactose and galactose to lactate, acetate, formate, and ethanol (mixed-acid fermentation) and fermented glucose to mainly lactate (homolactic fermentation). Strain H61 fermented glucose, lactose, and galactose to mainly lactate (homolactic fermentation). In strain G50, when growth efficiency was reduced by adding a metabolic inhibitor to the growth medium, SOD activity was higher than in the control; however, the metabolism was homofermentative. Aerobic conditions, but not glucose-limited conditions, increased SOD activity, and mixed-acid fermentation occurred. We conclude that the effect of carbohydrate on SOD activity in lactococci is strain dependent and that the activity of commercial lactococci can be enhanced through carbohydrate selection for mixed-acid fermentation or by changing the energy distribution, thus enhancing the value of the starter and the resulting dairy products.