A schematic representation of the srtB, fhu and hup regions of the L. monocytogenes chromosome shows loci involved in iron acquisition (red and blue genes are considered in this report). The srtB region (2.274 Mb) contains genes for secreted, peptidoglycan-associated proteins (lmo2185, 6), the sortase (srtB) that anchors them to the cell wall, a putative ABC-transporter (lmo2182–2184) and two SrtA-dependent cell wall proteins (lmo2178, 2179) (Bierne, 2004, Newton et al., 2005). The fhu region (2.031 Mb) encodes a substrate binding lipoprotein (FhuD) and a putative ferric hydroxamate ABC-transporter (FhuBCG) (Jin et al., 2005). The hupDGC locus (2.499 Mb) encodes a substrate binding lipoprotein (HupD) and a putative ABC transporter for Hn/Hb (Jin et al., 2005). Parenthetic values refer to the distance (bp) between adjacent genes.

Cells were grown in BHI overnight, and 2.5 × 10⁷ cells were subcultured into BHI containing 1 mM 2, 2’-bipyridyl. After 2 hours, Hn (2 mM in 40% DMSO) was diluted into the media to 0.2 µM. Growth curves defined by open symbols derive from parent cultures to which no Hn was added; curves from filled symbols represent data derived from cultures to which 0.2 µM Hn was added: (A) EGD-e, (X) ΔHupG, (Δ) ΔHupG_pPL2hupG⁺. For clarity, equivalent results with EGD-e derivatives carrying ΔhupC or ΔhupDGC are not shown. The plotted data represent a single experiment, but the experiment was repeated at least 3 times with the same results.

In panels A–C, wild type strain EGD-e (α), or its derivatives carrying the individual mutations Δhup (□); ΔsrtA (ϑ), ΔsrtB (Δ), ΔsrtAB (E) and Δlmo2185 (X) were grown in BHI, subcultured twice in MOPS-L, and tested for their ability to bind or transport [⁵⁹Fe]-Hn (Jin et al., 2005). Binding (A) and transport (B) assays revealed impaired Hn acquisition by Δhup, ΔsrtB, and Δlmo2185 at low nanomolar concentrations of the iron porphyrin, but no effect of Δlmo2185 at Hn concentrations ≥ 50 nM (C). In panel D, purified Lmo2185 was mixed with increasing concentrations of [⁵⁹Fe]-Hn and the protein was collected on filters, which were washed and counted to determine the amount of Hn bound (Experimental Procedures). The experiments were repeated 3 or 4 times; bars on the data points represent standard deviations of the mean values. In panel A, from the 6 fitted curves (bound vs total) the mean standard deviation was 6.7% for K_D values and 22.3% for capacity; in panel B, from the 5 fitted curves (enzyme kinetics) the mean standard deviatiion was 8.5% for K_M values and 20.7% for V_max; In panel D, from the fitted curve (bound vs total) the mean standard deviation was 4.1% for the K_D value and 18.7% for capacity.

L. monocytogenes 6H-FhuD and 6H-HupD were purified by affinity chromatography on Ni⁺⁺-NTA agarose and tested for their ability to bind ferric siderophores and Hn, by monitoring quenching of intrinsic Trp fluorescence. (Top panel) FhuD, the ferric hydroxamate binding protein, recognized a variety of iron complexes, including Hn, whereas (bottom panel) HupD was specific for its only ligand, Hn [(A) ferrioxamine B (FxB); (X) ferrichrome (Fc); (Δ) ferrichrome A (FcA); (E) ferric enterobactin (FeEnt); (α) haemin (Hn); (ϑ) protoporphyrin IX (PPIX)]. Removal of their 6H tags (by cleavage with thrombin) had no effect on the specificities or affinities of the binding proteins (Table 1).

EGD-e and its mutants were grown in BHI, subjected to iron deprivation at mid-log by addition of 1 mM bipyridyl, and plated on BHI agar containing 0.25 mM bipyridyl (Newton et al., 2005). (A) Hn/Hb utilization. Paper discs were placed on the agar, and 10µl aliquots of different iron compounds were applied to the discs: (1) 50 uM Fc; (2) 15 µM Hb; (3–5) 0.5 µM, 5 µM and 50 µM Hn, respectively. Mutants in the hup operon had reduced iron supply from Hn and Hb, whereas sortase mutants and Δlmo2185 showed little reduction in this assay. All the strains normally utilized the hydroxamate siderophore Fc. (B) Utilization of iron from purified Lmo2185. 10µl aliquots of compounds were applied with a micropipette tip directly to the agar: (1) 50 uM Fc; (2) 0.5 µM Hn; (3) 10.5 uM apo-Lmo2185; (4) partially saturated Lmo2185 (1.6 uM Lmo2185 containing 0.7 uM Hn). Both apo- and holo-Lmo2185 supplied iron to EGD-e, but not to its Δlmo2185 derivative.

A. Lmo2186 (Hbp1) and SauIsdC The sequences of LmoHbp1 and SauIsdC are 30% identical overall (69 of 227 residues), confirming their structural relatedness. The foundation of their conservation is in α and β secondary structure [the colored boxes in panels A and B correlate with the depiction of SauIsdC crystal structure (2O6P) in panel C]: in the α and β elements the extent of sequence identity is 78%. B. Lmo2185 (Hbp2), LmoHbp1 and SauIsdC. LmoHbp2 comprises a duplication of LmoHbp1, separated by ~200 amino acids (residues 163 – 351) in the middle of the protein. Its N-terminal half (a.a. 1 – 219) has 21% (43 of 207 residues) and 23% (51 of 227 residues) overall identity to LmoHbp1 and SauIsdC, respectively. The main sequence diversity occurs in loops and turns; in the α and β elements the extent of identity is 48% and 59%, respectively. Its C-terminal half (a.a. 352–569) has 25% (52 of 207 residues) and 22% (50 of 227 residues) overall identity to LmoHbp1 and SauIsdC, with 58% and 57% identity in α and β structure, respectively. C. SauIsdC. Eight residues its single α-helix and β-strands 7 and 8 complex heme in SauIsdC (2O6P). Theses amino acids in IsdC (yellow) are identical or conservatively substituted in Hbp1 and Hbp2 (both the N - and C - domains), including Y52 and Y132, which coordinate iron in the porphyrin ring. The only exception is I48 in IsdC, substituted by E59 in Hbp2. D and E. Sortase-dependent and independent heme transport through the Gram-positive bacterial cell envelope. The cytoplasmic membrane (phospholipids in stick format; phosphate atoms in space filling form) contains transporters for iron compounds, such as the HupDGC and FhuDBGC putative ABC-transporters (shown in ribbon format). The PG polymer (shown in wire format in panel D, and as space filling surfaces in panel E) surrounds the cell as a series of conjoined hexagonal pores with approximately 70 Å diameter (coordinates through the courtesy of S. Mobashery; Meroueh et al. 2006). This architecture creates pathways for diffusion of small solutes, like ferric siderophores (Fc) or porphyrins (Hn), through the PG matrix (best viewed in E). At concentrations above 50 nM, Hn permeates the PG layer and directly adsorbs to lipoproteins of the ABC-transporters. On the other hand, sortases A and B attach haem binding proteins (for example, listerial Hbp1 and Hbp2) and other proteins to the cell wall matrix by peptide bonds to Lys or DAP in PG. The anchored proteins create a sortase-dependent pathway of Hn/Hb (Hb (PDB file 3HF4) uptake. At very low Hn concentrations Hbp1 and Hbp2 initiate transport by capturing the iron porphyrin from solution and subsequently transferring it to the underlying ABC transporters. In this cartoon Hbp1 and Hbp2 were modeled from their relationship to IsdC (2O6P); HupDGC and FhuDBGC were modeled from their relationship to the iron ABC transporter of Haemophilus influenzae (2NQ2) and the maltose ABC transporter of E. coli (3FH6), respectively. The molecules were rendered and assembled with Chimera (UCSF); high resolution images with more detailed explanations are provided as supplementary Fig. S4).