Abstract
Nitric oxide (NO) is emerging as an important regulator of bacterial stress resistance, biofilm development, and virulence. One potential source of endogenous NO production in the pathogen Staphylococcus aureus is its NO-synthase (saNOS) enzyme, encoded by the nos gene. Although a role for saNOS in oxidative stress resistance, antibiotic resistance, and virulence has been recently-described, insights into the regulation of nos expression and saNOS enzyme activity remain elusive. To this end, transcriptional analysis of the nos gene in S. aureus strain UAMS-1 was performed, which revealed that nos expression increases during low-oxygen growth and is growth-phase dependent. Furthermore, nos is co-transcribed with a downstream gene, designated pdt, which encodes a prephenate dehydratase (PDT) enzyme involved in phenylalanine biosynthesis. Deletion of pdt significantly impaired the ability of UAMS-1 to grow in chemically-defined media lacking phenylalanine, confirming the function of this enzyme. Bioinformatics analysis revealed that the operon organization of nos-pdt appears to be unique to the staphylococci. As described for other S. aureus nos mutants, inactivation of nos in UAMS-1 conferred sensitivity to oxidative stress, while deletion of pdt did not affect this phenotype. The nos mutant also displayed reduced virulence in a murine sepsis infection model, and increased carotenoid pigmentation when cultured on agar plates, both previously-undescribed nos mutant phenotypes. Utilizing the fluorescent stain 4-Amino-5-Methylamino-2',7'-Difluorofluorescein (DAF-FM) diacetate, decreased levels of intracellular NO/reactive nitrogen species (RNS) were detected in the nos mutant on agar plates. These results reinforce the important role of saNOS in S. aureus physiology and virulence, and have identified an in vitro growth condition under which saNOS activity appears to be upregulated. However, the significance of the operon organization of nos-pdt and potential relationship between these two enzymes remains to be elucidated.
Highlights
Nitric Oxide (NO) is a highly reactive free radical gas that and rapidly diffuses across biological membranes [1,2]
This open reading frame (ORF) (SAR2008 of the MRSA252 genome, referred to as pdt) encodes a predicted prephenate dehydratase enzyme, which catalyzes the formation of phenylpyruvate from prephenate during phenylalanine biosynthesis [23]
Our current results have corroborated the contribution of Staphylococcus aureus is its NO-synthase (saNOS) to hydrogen peroxide resistance in the MSSA strain UAMS-1, and have illustrated that carotenoid pigmentation is increased in a nos mutant when grown on tryptic soy agar (TSA) plates (Fig. 6A), a phenotype that has not been previously described for S. aureus nos mutants
Summary
Nitric Oxide (NO) is a highly reactive free radical gas that and rapidly diffuses across biological membranes [1,2]. The role of bacterial NOS can be quite varied, participating in diverse physiological functions such as biofilm development, biosynthesis of plant phytotoxins, stress resistance and virulence [12,13,14,15,16,17,18]. This versatility suggests a specificity of function to individual bacterial species, an increased understanding of the regulation and exact cellular targets of bacterial NOS may help divulge an evolutionary link between prokaryotic and eukaryotic organisms. Much less is understood about the regulation of bacterial NOS enzyme expression, activity, and participation in cell signaling
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