title: Functional profiling in Streptococcus mutans: construction and examination of a genomic collection of gene deletion mutants.
abstract: A collection of tagged deletion mutant strains was created in Streptococcus mutans UA159 to facilitate investigation of the aciduric capability of this oral pathogen. Gene-specific barcoded deletions were attempted in 1432 open reading frames (representing 73% of the genome), and resulted in the isolation of 1112 strains (56% coverage) carrying deletions in distinct non-essential genes. As S. mutans virulence is predicated upon the ability of the organism to survive an acidic pH environment, form biofilms on tooth surfaces, and out-compete other oral microflora, we assayed individual mutant strains for the relative fitness of the deletion strain, compared with the parent strain, under acidic and oxidative stress conditions, as well as for their ability to form biofilms in glucose- or sucrose-containing medium. Our studies revealed a total of 51 deletion strains with defects in both aciduricity and biofilm formation. We have also identified 49 strains whose gene deletion confers sensitivity to oxidative damage and deficiencies in biofilm formation. We demonstrate the ability to examine competitive fitness of mutant organisms using the barcode tags incorporated into each deletion strain to examine the representation of a particular strain in a population. Co-cultures of deletion strains were grown either in vitro in a chemostat to steady-state values of pH 7 and pH 5 or in vivo in an animal model for oral infection. Taken together, these data represent a mechanism for assessing the virulence capacity of this pathogenic microorganism and a resource for identifying future targets for drug intervention to promote healthy oral microflora.
authors.affiliation: Department of Microbiology and Immunology, University of Rochester, Rochester, NY, USA.|Center for Oral Biology, University of Rochester, Rochester, NY, USA.
authors.forename: R G
authors.initials: RG
authors.lastname: Quivey
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester, Rochester, NY, USA.
authors.forename: E J
authors.initials: EJ
authors.lastname: Grayhack
authors.affiliation: Center for Oral Biology, University of Rochester, Rochester, NY, USA.
authors.forename: R C
authors.initials: RC
authors.lastname: Faustoferri
authors.affiliation: Center for Oral Biology, University of Rochester, Rochester, NY, USA.
authors.forename: C J
authors.initials: CJ
authors.lastname: Hubbard
authors.affiliation: Department of Microbiology and Immunology, University of Rochester, Rochester, NY, USA.
authors.forename: J D
authors.initials: JD
authors.lastname: Baldeck
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester, Rochester, NY, USA.
authors.forename: A S
authors.initials: AS
authors.lastname: Wolf
authors.affiliation: Center for Oral Biology, University of Rochester, Rochester, NY, USA.
authors.forename: M E
authors.initials: ME
authors.lastname: MacGilvray
authors.affiliation: Center for Oral Biology, University of Rochester, Rochester, NY, USA.
authors.forename: P L
authors.initials: PL
authors.lastname: Rosalen
authors.affiliation: Center for Oral Biology, University of Rochester, Rochester, NY, USA.
authors.forename: K
authors.initials: K
authors.lastname: Scott-Anne
authors.affiliation: Center for Oral Biology, University of Rochester, Rochester, NY, USA.
authors.forename: B
authors.initials: B
authors.lastname: Santiago
authors.affiliation: Department of Biological Sciences, Rochester Institute of Technology, Rochester, NY, USA.
authors.forename: S
authors.initials: S
authors.lastname: Gopal
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester, Rochester, NY, USA.
authors.forename: J
authors.initials: J
authors.lastname: Payne
authors.affiliation: Department of Microbiology and Immunology, University of Rochester, Rochester, NY, USA.
authors.forename: R E
authors.initials: RE
authors.lastname: Marquis
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D018441:Biofilms / Q000254:growth & development; D058893:DNA Barcoding, Taxonomic; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D056084:Genetic Fitness; D016680:Genome, Bacterial; D023281:Genomics; D006863:Hydrogen-Ion Concentration; D009055:Mouth / Q000382:microbiology; D009154:Mutation; D018384:Oxidative Stress / Q000235:genetics; D051381:Rats; D013295:Streptococcus mutans / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity
chemical_list: D001426:Bacterial Proteins
keywords: genomics; pathogenesis; physiology