title: Contribution of the nos-pdt operon to virulence phenotypes in methicillin-sensitive Staphylococcus aureus.
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.
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, United States of America.
authors.forename: April M
authors.initials: AM
authors.lastname: Sapp
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Austin B
authors.initials: AB
authors.lastname: Mogen
authors.affiliation: Department of Microbiology, North Carolina State University, Raleigh, North Carolina, United States of America.
authors.forename: Erin A
authors.initials: EA
authors.lastname: Almand
authors.affiliation: Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, Florida, United States of America.
authors.forename: Frances E
authors.initials: FE
authors.lastname: Rivera
authors.affiliation: Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, Florida, United States of America.
authors.forename: Lindsey N
authors.initials: LN
authors.lastname: Shaw
authors.affiliation: Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Richardson
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Kelly C
authors.initials: KC
authors.lastname: Rice
mesh_terms: D000818:Animals; D002338:Carotenoids / Q000378:metabolism; D004195:Disease Models, Animal; D005260:Female; D005452:Fluoresceins / Q000378:metabolism; D005798:Genes, Bacterial; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008712:Methicillin / Q000494:pharmacology*; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics*; D009876:Operon / Q000235:genetics*; D018384:Oxidative Stress / Q000187:drug effects; D010641:Phenotype; D010649:Phenylalanine / Q000494:pharmacology; D010858:Pigmentation / Q000187:drug effects; D011302:Prephenate Dehydratase / Q000235:genetics*; D026361:Reactive Nitrogen Species / Q000378:metabolism; D018805:Sepsis / Q000382:microbiology / Q000473:pathology; D013211:Staphylococcus aureus / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity*; D016019:Survival Analysis; D014158:Transcription, Genetic / Q000187:drug effects; D014774:Virulence / Q000187:drug effects / Q000235:genetics
chemical_list: D005452:Fluoresceins; D026361:Reactive Nitrogen Species; C029569:diacetyldichlorofluorescein; D009569:Nitric Oxide; D002338:Carotenoids; D010649:Phenylalanine; D019001:Nitric Oxide Synthase; D011302:Prephenate Dehydratase; D008712:Methicillin