In addition to the importance of heme as

In addition to the importance of heme as an enzymatic cofactor, heme also acts as an important iron source. In order to access the iron within heme, S. aureus encodes heme degrading enzymes known as heme oxygenases (Skaar et al., 2004b; Wu et al., 2005). The two heme oxygenases encoded by S. aureus, IsdG and IsdI, are essential for growth under iron deplete conditions where heme is the sole source of iron (Fig. 1). We initially sought to investigate the role of heme degradation products in S. aureus. However, upon performing RNA-Sequencing analysis, we identified that expressing isdI under the constitutively expressed lgt promoter led to an increase in expression of genes linked to oxygen-independent URMC-099 production (Fig. 2). Since the cultures for these experiments had been grown aerobically, we hypothesized that the overproduction of isdI led to excess heme degradation, resulting in an insufficient amount of heme present to populate the cytochromes required for efficient aerobic respiration. To test this hypothesis, we developed strains of S. aureus expressing each heme oxygenase either under the endogenous promoters (ΔisdGI pisd.) or the constitutively expressed lgt promoter (ΔisdGI plgt.). When these strains were grown under iron limitation with heme as the sole source of iron, the endogenously expressed heme oxygenases grew better than the constitutively expressed heme oxygenases (Fig. 3B). We next investigated the effect of heme oxygenase transcriptional regulation on cellular heme abundance and found that the strains constitutively expressing isdI and isdG contained less heme (Fig. 4A). Additionally, when the fur gene is removed from S. aureus, there is also a significant decrease in cellular heme levels compared to the strain lacking heme oxygenase activity (Fig. 4B). As a result of this decrease in cellular heme, the strains constitutively expressing the heme oxygenases produce higher levels of the fermentation end-product lactate (Fig. 5A). Additionally, the constitutively isdI expressing strain is also more resistant to gentamicin, which requires active electron transport for entry into the cell (Fig. 5B). Finally, the constitutively expressing heme oxygenase strains had a slight growth defect compared to a strain lacking heme oxygenase activity when grown on the non-fermentable carbon source glycerol (Fig. 5C). S. aureus strains that are unable to respire, due to lack of either heme or menadione, exhibit a small colony variant (SCV) phenotype (Bates et al., 2003; von Eiff et al., 2006). These SCVs have increased resistance to aminoglycoside antibiotics that contributes to their ability to cause persistent infections (Musher et al., 1979; Kahl et al., 1998). While constitutively expressing heme oxygenases in strains of S. aureus do not have the same colony morphology as SCVs, the dysregulation of the heme oxygenases leads to a significant decrease in cellular heme. The reduced heme levels exhibited by the S. aureus strains constitutively expressing heme oxygenases lower overall fitness. Therefore, connecting heme oxygenase expression to iron availability through Fur may ensure optimal heme levels for bacterial fitness. This is especially important since removing fur leads to a significant decrease in heme levels compared to a strain lacking heme oxygenase activity. Additionally, all functionally identified IsdG family heme oxygenases are regulated by Fur, as are the heme acquisition systems encoded within these organisms. We predict that heme oxygenases are co-expressed with heme acquisition systems to prevent the heme oxygenases from degrading the endogenously biosynthesized heme. We anticipate that this work will be broadly applicable within bacteria that contain Fur-regulated heme oxygenases, since removing fur leads to a decrease in cellular heme. This could have extensive physiological implications on not only respiration, but also on a variety of other heme-associated processes, such as catalase functionality and bacterial nitric oxide synthase.