Physiological characterization of Haemophilus influenzae Rd deficient in its glutathione-dependent peroxidase PGdx.
The chimeric peroxidase PGdx of Haemophilus influenzae Rd belongs to a recently identified family of thiol peroxidases capable of reducing hydrogen peroxide as well as alkylhydroperoxides by means of glutathione redox cycling. In the present study, we constructed a H. influenzae Rd strain, deficient in its PGdx encoding gene (open reading frame HI0572). The mutant was shown by disk inhibition and liquid culture growth assays to exhibit increased susceptibility to organic hydroperoxides. The hampered growth was restored by complementing the interrupted gene on the genome with a replicating plasmid bearing an intact copy of the gene, hereby rejecting the possible influences of polar effects. Elevated levels of hydrogen peroxide scavenging activity, due to the catalase HktE, were measured in the absence of a functional pgdx gene rendering the mutant more resilient against hydrogen peroxide. On the other hand, after initiation of the stationary phase, aerobic cultures of the pgdx mutant were practically devoid of living cells, whereas wild-type counterparts retained viability. This observed feature was alleviated by complementation with the functional gene or with the addition of catalase.
Pauwels
Frederik
F
Laboratory of Protein Biochemistry and Protein Engineering, Ghent University, K. L. Ledeganckstraat 35, 9000 Ghent, Belgium.
Vergauwen
Bjorn
B
Van Beeumen
Jozef J
JJ
D015139:Blotting, Southern; D015153:Blotting, Western; D002374:Catalase / Q000737:chemistry / Q000378:metabolism; D002455:Cell Division; D004058:Diffusion; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D005838:Genotype; D006193:Haemophilus influenzae / Q000201:enzymology*; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D009154:Mutation; D016366:Open Reading Frames; D009195:Peroxidase / Q000235:genetics / Q000502:physiology*; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D013053:Spectrophotometry; D013997:Time Factors; D014466:Ultraviolet Rays; D015854:Up-Regulation; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
D020122:tert-Butylhydroperoxide; D006861:Hydrogen Peroxide; D010544:Peroxidases; D002374:Catalase; C484690:HktE protein, Haemophilus influenzae; C484691:PGdx protein, Haemophilus influenzae; D009195:Peroxidase