title: Signature-tagging of a bacterial isolate demonstrates phenotypic variability of the progeny in vivo in the absence of defined mutations.
abstract: Awareness of the high degree of redundancy that occurs in several nutrient uptake pathways of Haemophilus influenzae led us to attempt to develop a quantitative STM method that could identify both null mutants and mutants with decreased fitness that remain viable in vivo. To accomplish this task we designed a modified STM approach that utilized a set of signature tagged wild-type (STWT) strains (in a single genetic background) as carriers for mutations in genes of interest located elsewhere in the genome. Each STWT strain differed from the others by insertion of a unique, Q-PCR-detectable, seven base pair tag into the same redundant gene locus. Initially ten STWTs were created and characterized in vitro and in vivo. As anticipated, the STWT strains were not significantly different in their in vitro growth. However, in the chinchilla model of otitis media, certain STWTs outgrew others by several orders of magnitude in mixed infections. Removal of the predominant STWT resulted in its replacement by a different predominant STWT on retesting. Unexpectedly we observed that the STWT exhibiting the greatest proliferation was animal dependent. These findings identify an inherent inability of the signature tag methodologies to accurately elucidate fitness in this animal model of infection and underscore the subtleties of H. influenzae gene regulation.
authors.affiliation: Department of Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. pwhitby@ouhsc.edu
authors.forename: Paul W
authors.initials: PW
authors.lastname: Whitby
authors.forename: Timothy M
authors.initials: TM
authors.lastname: VanWagoner
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Morton
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Seale
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Springer
authors.forename: Randy J
authors.initials: RJ
authors.lastname: Hempel
authors.forename: Terrence L
authors.initials: TL
authors.lastname: Stull
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002682:Chinchilla; D005821:Genetic Techniques; D006192:Haemophilus Infections / Q000382:microbiology*; D006193:Haemophilus influenzae / Q000235:genetics* / Q000254:growth & development* / Q000302:isolation & purification; D050296:Microbial Viability; D009154:Mutation; D010033:Otitis Media / Q000382:microbiology*; D010641:Phenotype
chemical_list: D001426:Bacterial Proteins