title: Development of a noninvasive reporter system for gene expression in Porphyromonas gingivalis.
abstract: Several reports have supported the association of Porphyromonas gingivalis with periodontal disease. Genetic studies are vital for understanding the relative importance of virulence factors in this organism. Thus, gene reporters may prove useful for the study of gene expression in this organism. We have investigated the use of the green fluorescent protein (GFP), bacterial luciferase, and bifunctional xylosidase/arabinosidase enzyme (XA) as reporters of gene expression in P. gingivalis. Fusion cassettes containing the promoterless tetracycline resistant gene [tetA(A)Q2] and the promoterless gfp, luxAB, or xa gene were placed under the control of the rgpA promoter in P. gingivalis W83 using recombinational allelic exchange. The rgpA gene encodes for an arginine-specific protease in P. gingivalis. No GFP activity was detected in P. gingivalis isogenic mutants carrying the rgpA::gfp-tetA(Q)2 fusion construct. Luciferase activity in P. gingivalis mutants carrying the rgpA::luxAB-tetA(Q)2 fusion was only detected in the presence of exogenous FMNH(2). xa gene expression in P. gingivalis with the rgpA::xa-tetA(Q)2 fusion construct was detected in crude extracts using rho-nitrophenol derivatives as substrate and on agar plates with methylumbelliferyl derivatives under long-wave ultraviolet light. This indicates that both luxAB and xa genes can be used as reporters of gene expression in P. gingivalis. However, only the xa gene can be used as a noninvasive reporter gene.
authors.affiliation: Department of Microbiology and Molecular Genetics, School of Medicine, Loma Linda, California 92350, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Liu
authors.forename: H
authors.initials: H
authors.lastname: Abaibou
authors.forename: H M
authors.initials: HM
authors.lastname: Fletcher
mesh_terms: D019677:Artificial Gene Fusion; D015153:Blotting, Western; D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D004247:DNA / Q000032:analysis / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter / Q000235:genetics*; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D016966:Porphyromonas gingivalis / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014995:Xylosidases / Q000235:genetics* / Q000378:metabolism
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; D008156:Luciferases; D006026:Glycoside Hydrolases; D014995:Xylosidases; C019713:alpha-N-arabinofuranosidase