Expression of the tpr protease gene of Porphyromonas gingivalis is regulated by peptide nutrients.
The Tpr protease of Porphyromonas gingivalis W83 is a membrane-associated enzyme capable of hydrolyzing chromogenic substrates for trypsin and bacterial collagenases. A previous study by us indicated that Tpr expression was increased under conditions of nutrient limitation. In the present study, we further characterized expression of the tpr gene using a tpr::lacZ reporter gene construct under a range of nutrient conditions. In P. gingivalis, transcription of tpr was initiated 215 bp upstream of the coding region and regulation of tpr expression was at the level of transcription. Deletion mutations in the tpr upstream region identified the promoter region immediately upstream of the transcription start site, determined by primer extension analysis. Three identical 17-bp direct repeats identified within the 5' end of tpr mRNA were involved in tpr regulation. In an Escherichia coli background, tpr transcription was initiated after an AT-rich region upstream of tpr but not at the P. gingivalis start site. Tpr expression in P. gingivalis was suppressed by the addition of peptide and protein nutrients to a peptide-limited growth medium but was only slightly affected by addition of free amino acids. Low-molecular-weight fractions of brain heart infusion rich in phenylalanine, proline, and alanine had the greatest inhibitory effects on expression of the tpr::lacZ construct. Addition of the dipeptide phenylalanyl-phenylalanine to the growth medium resulted in a 10-fold decrease in tpr expression. This suggests that specific phenylalanine-containing peptides are a major factor controlling Tpr expression. Neither hemin starvation, heat shock, nor pH change had significant effects on Tpr expression.
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3.
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McBride
D001483:Base Sequence; D018387:Codon, Initiator; D010450:Endopeptidases / Q000096:biosynthesis* / Q000235:genetics*; D004789:Enzyme Activation / Q000187:drug effects; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D005798:Genes, Bacterial / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000494:pharmacology*; D016966:Porphyromonas gingivalis / Q000235:genetics* / Q000254:growth & development; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014158:Transcription, Genetic; D014162:Transfection; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D018387:Codon, Initiator; D010455:Peptides; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase; D010450:Endopeptidases