Physiological Characterization of a Heme-Deficient Mutant of Staphylococcus aureus by a Proteomic Approach

Abstract

The high-resolution two-dimensional (2D) protein gel electrophoresis technique combined with matrixassisted laser desorption ionization-time of flight mass spectrometry was used for identification of proteins whose levels were changed by a mutation in hemB. Cytoplasmic protein extracts obtained from the mutant and the wild type (strain COL) at different stages of growth in tryptone soya broth (exponential, transitional, and stationary growth phases) were separated on 2D protein gels. Comparison of the 2D patterns of the protein extracts of the two strains revealed major differences. Because the electron transport chain of the mutant is interrupted due to the deficiency of heme, this organism should be unable to use oxygen or nitrate as a terminal electron acceptor. Consistent with this hypothesis, proteins involved in the glycolytic pathway and related pathways (glyceraldehyde-3-phosphate dehydrogenase, enolase, and phosphoglycerate kinase) and in fermentation pathways (lactate dehydrogenase, alcohol dehydrogenase, and pyruvate formate lyase) were induced in exponentially growing cells of the mutant. These results strongly indicate that the hemB mutant generates ATP from glucose or fructose only by substrate phosphorylation. Analyses of the fermentation reactions showed that the main product was lactate. Although pyruvate formate lyase (Pfl) and pyruvate dehydrogenase were present, neither ethanol nor acetate was detected in significant amounts. Presumably, Pfl was not activated in the presence of oxygen, and pyruvate dehydrogenase might have very low activity. Transcriptional analysis of citB, encoding the aconitase, revealed that the activity of the citrate cycle enzymes was down-regulated in the hemB mutant. The arginine deiminase pathway was also induced, and it could provide ATP as well. Furthermore, the amounts of most of the extracellular virulence factors were significantly reduced by a mutation in hemB, which is consistent with previous reports.

Proteomics Analyses ofBacillus subtilisafter Treatment with Plumbagin, a Plant-Derived Naphthoquinone

“…Moreover, the induction of nitrate reductase alpha and nitrate reductase beta chain strongly indicates the anaerobic respiration (Cortial et al, 2010;Kohler et al, 2003;Ye et al, 2000) and induction of anaerobic marker protein YwfI suggests that the anaerobic respiration was dominated under plumbagin treatment . In addition, nitroreductases involved in oxidation of NADH and scavengers of peroxides were induced.…”

“…Our results indicate that plumbagin treatment significantly repressed TCA and the electron transport chain, which indicates the blockage in energy generation. Alternatively, glycolysis, which can provide the energy via substrate level phosphorylation, anaerobic respiration or fermentative process to support the growth, was found to be induced due to plumbagin treatment (Cruz Ramos et al, 2000;Kohler et al, 2003) (Fig. 5).…”

Immunomodulation of Nasal Epithelial Cells by Staphylococcus aureus-Derived Serine Proteases

“…The sitedirected hemB mutants represent a stable and genetically defined SCV phenotype (18,20,21), and mutant III33 has been shown to produce almost 20 times more protease than its parental strain (16).…”

At the Crossroads of Bacterial Metabolism and Virulence Factor Synthesis in Staphylococci

“…Protons return to the cytoplasm by way of the F o subunit of the F o F 1 ATP synthase complex and drive the dissociation of newly formed ATP. The inactivation of the electron transfer chain, either by genetic inactivation (63,124) or by extreme iron restriction (62), results in a growth phenotype similar to that of anaerobic/fermentative growth. The inactivation of the electron transfer chain produces a clinically relevant phenotype in staphylococci known as the small-colony variant phenotype.…”