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Construction and characterization of tpr and prT single and double mutants of Porphyromonas gingivalis W83 Wan, Steven


DNA and amino acid sequence data show remarkable homologies between many Arg-X active proteases produced by P. gingivalis. Among the proteases sequenced thus far, only the Tpr and PrtT do not exhibit significant homologies to any other Arg-X proteases or with each other. This finding suggested that Tpr and PrtT might play unique roles in the growth and metabolism of P. gingivalis. In this investigation, we analyzed the effects of allelic replacement mutagenesis of prtT, tpr and both prtT and tpr on growth, proteolytic activity and hemagglutination of P. gingivalis in media of varying nutritional composition. Comparison of wild type and mutant strains revealed that the growth rates of prtT'mutants were more significantly reduced in nutritionally rich (BSA-supplemented) media than in nutritionally poor (gelatin-supplemented) media. The growth rates of the tpr mutants were more significantly reduced in poorer media than in richer media. The Arg-X (BAPNA-hydrolyzing) activity of wild type and mutant strains was nutrient- and growth phase-dependent. The prtT mutants produced lower protease activity in all protein-supplemented media during early-log phase, but levels similar to that of wild type during late-log phase. The tpr mutants produced lower protease activity during initial phases of growth in protein supplemented media as well as in nutritionally poor media during late-log phase. The relative magnitude of hemagglutination activity reduction for Tpr- and PrtT-deficient mutants was directly proportional to the relative level of reduction in BAPNA-hydrolyzing activity during initial phases of growth. Quantitative messenger RNA studies showed that prtT mRNA was expressed at a higher level during early-log phase in nutritionally rich media, and that the tpr mRNA was expressed at a higher level in nutritionally poor media in late-log phase. These results suggest that PrtT protease may play an important role during early-log phase growth in nutritionally rich (BSAsupplemented) media and Tpr may be important during late-log phase growth in nutritionally poor (gelatin-supplemented) media. Both may play a direct or indirect roles in hemagglutination.

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