A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus

Abstract

Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of ironregulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD-I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis.

The SaeRS Two-Component System Is a Direct and Dominant Transcriptional Activator of Toxic Shock Syndrome Toxin 1 in Staphylococcus aureus

“…Equal amounts of forward and reverse DNA oligonucleotides (20 M) were heated to 100°C for 3 min and allowed to anneal. Phosphorylation of SaeR and the EMSA were done as previously described (25,28), with some modifications. Proteins and DNA probes were incubated at room temperature in a 25-l volume containing phosphorylation buffer (10 mM Tris-HCl [pH 7.4], 50 mM KCl, 5 mM MgCl 2 , 10% glycerol), 240 g/ml of bovine serum albumin, and 12 g/ml poly (dI-dC).…”

Heme Synthesis and Acquisition in Bacterial Pathogens

“…Heme is the preferred iron-source during systemic infection, in part because a heme-responsive transcriptional regulator activates iron siderophore synthesis only when heme-iron is unavailable [84,122]. The role of the Isd system has been extensively demonstrated in murine infection models.…”

Metal homeostasis in infectious disease: recent advances in bacterial metallophores and the human metal-withholding response

“…A sbnI -deficient mutant strain of S. aureus exhibited a severe growth defect under iron-limited growth conditions [10]; however, enzymology studies revealed that SbnI is not required for the in vitro biosynthesis of SB [40], and no evidence for a role of sbnI in SB transport was found. Indeed, recent investigations demonstrated that SbnI is a regulatory protein.…”

“…Indeed, recent investigations demonstrated that SbnI is a regulatory protein. It binds DNA upstream of sbnD and is required for the expression of SbnD and the biosynthetic enzymes SbnEFGH (Figure 3B) [10]. Further interrogation revealed that SbnI binds heme, and that formation of the SbnI-heme complex prevents the SbnI-DNA interaction.…”

“…We describe the discovery of staphylopine [9], a metallophore that S. aureus employs for acquiring multiple divalent first-row transition metals. Next, we present a link between staphylococcal heme uptake and the biosynthesis of staphyloferrin B, a high-affinity iron chelator or siderophore that is implicated in the virulence of S. aureus [10]. In the third vignette, we discuss enterobactin (Ent), a siderophore of Escherichia coli , and describe work that probes the interplay among Ent, other iron-chelating metabolites, and the human host-defense protein lipocalin-2 (LCN2) [11].…”