(134b) Elucidating the Physiological Significance of Nitric Oxide Synthase (NOS) in Staphylococcus aureus through Metabolic Modeling

In recent years, whole genome sequencing has accelerated the reconstruction of genome scale metabolic models of several species. However, often these models do not accurately predict biological phenotypes due to inherent insufficiencies including metabolic gaps, unbalanced reactions and lack of key physiological information. In the present study, the genome-scale metabolic model of the bacterial pathogen, Staphylococcus aureus was reconstructed from ModelSEED and previous strain-specific models^1,2. Reactions were elemental and charge balanced. Upon manual and automated gap-filling the model has 842 metabolic genes, 1430 metabolites and 1701 reactions including transport and export reactions. The model was applied to understanding the physiological function of S. aureus nitric oxide synthase (NOS).

The S. aureus NOS is an enzyme that converts arginine to citrulline and produces the highly reactive nitric oxide radical (NO·) as a byproduct. Under aerobic conditions, NO· is further decomposed to nitrates and nitrites. Inactivation of nos decreases growth yields. However, the mechanism by which NOS activity enhances staphylococcal biomass production is unclear. Nuclear magnetic resonance (NMR) spectroscopic analysis of the nos mutant revealed decreased TCA cycle activity compared to its isogenic wild-type strain. It is conceivable that carbon-flux of arginine to 2-oxoglutarate through the glutamate node following NOS activity may be important for maintenance of TCA cycle activity. Alternately, our results also support a role for nitrites that indirectly result from NOS activity in stimulating terminal cytochrome (quinol oxidase) function and enhancing rate of respiration. The latter may also affect TCA cycle activity and biomass. Our analysis using the reconstructed genome scale metabolic model suggests that the latter possibility wherein nitrites affect respiration is more likely to modulate TCA cycle activity given that carbon flux through NOS is limited.

We have directly confirmed this prediction of the metabolic model by demonstrating that biomass production is rescued in the nos mutant following restoration of respiration by the addition of physiological concentrations of nitrite in the growth media. Finally, addition of 2-oxoglutarate (a TCA cycle metabolite) to the media does not restore biomass of the nos mutant to wild-type levels even though it is completely consumed by these strains. This suggests that carbon flux to the TCA cycle through the glutamate node is not a significant contributor of biomass in the nos mutant. These findings not only attest to the predictive power of our metabolic model but also highlight the biological role of bacterial NOS as an activator of quinol oxidase mediated aerobic respiration.

References:

1. Becker SA, Palsson BO. Genome-scale reconstruction of the metabolic network in Staphylococcus aureus N315: an initial draft to the two-dimensional annotation. BMC microbiology. Mar 07 2005;5:8.

2. Bosi E, Monk JM, Aziz RK, Fondi M, Nizet V, Palsson BO. Comparative genome-scale modelling of Staphylococcus aureus strains identifies strain-specific metabolic capabilities linked to pathogenicity. Proc Natl Acad Sci U S A. Jun 28 2016;113(26):E3801-3809.