T Choudhury1, E F Sato, M Inoue. 1. Department of Biochemistry & Molecular Pathology, Osaka City University Medical School, Abeno, Osaka, Japan.
Abstract
BACKGROUND/AIMS: The mechanisms of nitric oxide (NO) production by bacteria in the oral cavity are still not clearly defined but salivary streptococci have been reported to generate NO. The aim of this study was to clarify the mechanism of nitrite metabolism and generation of NO by Streptococcus mutans, a major pathogen of dental caries. METHODS: We searched the genomic database of oral pathogens for nitrite reductase and used a polymerase chain reaction (PCR) to clone the nirJ gene from S. mutans GS5. His-tagged recombinant NirJ protein was expressed in Escherichia coli BL21 and characterized. We constructed a nirJ gene-disrupted mutant strain of S. mutans (DeltanirJ) to analyze the physiological significance of nirJ. RESULTS: S. mutans generates NO from nitrite, probably as a result of the possession of nitrite reductase. We cloned the nirJ gene from S. mutans GS5 by PCR. The recombinant NirJ protein catalyzed the reduction of nitrite with a K(m) value of 3.37 microM and a specific activity of 2.5 micromol/min/mg of protein at 37 degrees C. Biochemical analysis revealed that the nitrite-reducing activity of the mutant (DeltanirJ) strain was significantly lower than that of the wild-type strain. The growth of the mutant strain, but not of the wild-type strain, was strongly suppressed by the presence of physiological levels of nitrite ( approximately 0.2 mM) in saliva. CONCLUSION: These observations suggest that the elimination of nitrite and/or the generation of NO are important for the survival of S. mutans in the oral cavity.
BACKGROUND/AIMS: The mechanisms of nitric oxide (NO) production by bacteria in the oral cavity are still not clearly defined but salivary streptococci have been reported to generate NO. The aim of this study was to clarify the mechanism of nitrite metabolism and generation of NO by Streptococcus mutans, a major pathogen of dental caries. METHODS: We searched the genomic database of oral pathogens for nitrite reductase and used a polymerase chain reaction (PCR) to clone the nirJ gene from S. mutans GS5. His-tagged recombinant NirJ protein was expressed in Escherichia coli BL21 and characterized. We constructed a nirJ gene-disrupted mutant strain of S. mutans (DeltanirJ) to analyze the physiological significance of nirJ. RESULTS:S. mutans generates NO from nitrite, probably as a result of the possession of nitrite reductase. We cloned the nirJ gene from S. mutans GS5 by PCR. The recombinant NirJ protein catalyzed the reduction of nitrite with a K(m) value of 3.37 microM and a specific activity of 2.5 micromol/min/mg of protein at 37 degrees C. Biochemical analysis revealed that the nitrite-reducing activity of the mutant (DeltanirJ) strain was significantly lower than that of the wild-type strain. The growth of the mutant strain, but not of the wild-type strain, was strongly suppressed by the presence of physiological levels of nitrite ( approximately 0.2 mM) in saliva. CONCLUSION: These observations suggest that the elimination of nitrite and/or the generation of NO are important for the survival of S. mutans in the oral cavity.