Insertional Mutation of ftsH Gene in Streptococcus pneumoniae Causes Stress Sensitivities

FtsH is a membrane-bound, ATP-dependent protease involved in various cellular functions. To understand its roles in Streptococcus pneumoniae and host-pathogen interactions, we inactivated the ftsH gene of D39 strain by inserting a tetracycline-resistance (tet) gene. Several recombinants containing the tet cassette within the ftsH gene were confirmed by Western immunoblotting for the absence of pneumococcal FtsH protein that could cross-react with antiserum raised against Escherichia coli FtsH. Compared with the wild-type D39 strain, the ftsH null mutants grew slowly with encapsulation and alpha-hemolysis on blood agar plates, but failed to grow in liquid media other than Todd Hewitt yeast extract broth. Even fresh cultures of ftsH null mutants appeared gram-negative. When the incubation temperature of liquid cultures was shifted from 37degrees C to 40degrees C, the mutants gradually lysed, whereas the shift to 30degrees C abolished further growth. The mutants also exhibited increased sensitivity to salt and remarkable growth inhibition by optochin. These observations suggest that no functional FtsH protein in pneumococcal cells causes a loss of cell surface integrity, resulting in impairment of cell growth under normal and stressful conditions.

Insertional Mutation of ftsH Gene in Streptococcus pneumoniae Causes Stress Sensitivities

FtsH is a membrane-bound, ATP-dependent protease involved in various cellular functions. To understand its roles in Streptococcus pneumoniae and host-pathogen interactions, we inactivated the ftsH gene of D39 strain by inserting a tetracycline-resistance (tet) gene. Several recombinants containing the tet cassette within the ftsH gene were confirmed by Western immunoblotting for the absence of pneumococcal FtsH protein that could cross-react with antiserum raised against Escherichia coli FtsH. Compared with the wild-type D39 strain, the ftsH null mutants grew slowly with encapsulation and alpha-hemolysis on blood agar plates, but failed to grow in liquid media other than Todd Hewitt yeast extract broth. Even fresh cultures of ftsH null mutants appeared gram-negative. When the incubation temperature of liquid cultures was shifted from 37degrees C to 40degrees C, the mutants gradually lysed, whereas the shift to 30degrees C abolished further growth. The mutants also exhibited increased sensitivity to salt and remarkable growth inhibition by optochin. These observations suggest that no functional FtsH protein in pneumococcal cells causes a loss of cell surface integrity, resulting in impairment of cell growth under normal and stressful conditions.