TCA cycle activity in Staphylococcus aureus is essential for iron-regulated synthesis of staphyloferrin A, but not staphyloferrin B: the benefit of a second citrate synthase.

Staphylococcus aureus elaborates two citrate-containing siderophores, staphyloferrin A (SA) and staphyloferrin B (SB), that enhance growth under iron-restriction, yet, paradoxically, expression of the TCA cycle citrate synthase, CitZ, is downregulated during iron starvation. Iron starvation does, however, result in expression of SbnG, recently identified as a novel citrate synthase that is encoded from within the iron-regulated SB biosynthetic locus, suggesting an important role for SbnG in staphyloferrin production. We demonstrate that during growth of S. aureus in iron-restricted media containing glucose, SB is produced but, in contrast, SA production is severely repressed; accordingly, SB-deficient mutants grow poorly in these media. Hypothesizing that reduced TCA cycle activity hinders SA production, we show that a citZ mutant is capable of SB synthesis, but not SA synthesis, providing evidence that SbnG does not generate citrate for incorporation into SA. A citZ sbnG mutant synthesizes neither staphyloferrin, is severely compromised for growth in iron-restricted media, and is significantly more impaired for virulence than either of the single-deletion mutants. We propose that SB is the more important of the two siderophores for S. aureus insofar as it is synthesized, and supports iron-restricted growth, without need of TCA cycle activity.