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3.4.1 General Properties of Heme ATP Binding Cassette Transporters. Heme iron can be transported into the cell via heme ATP binding cassette (ABC) transporters. These transporters utilize the energy of ATP to transport Hm into a cell (Higgins & Linton, 2001; Koster, 2001; Clarke et al., 2001; Stojiljkovic & Perkins-Balding, 2002; Higgins & Linton, 2004; Davidson & Chen, 2004; Jones & George, 2004; Linton & Higgins, 2007; Stieger & Higgins, 2007). They are composed of a heme binding protein (HBP), a membrane permease, and an ATPase (Figure 3.2). An individual bacterial heme ABC operon typically codes for one HBP, and usually only one permease and one ATPase, though there are exceptions (Perkins-Balding et al., 2004).

The HBP is generally membrane-anchored in Gram positive bacteria and periplasmic in Gram negative bacteria; sequence homology can be found between both types of bacterial HBPs. Most HBPs consist of two core domains bridged by a flexible linker. Quiocho and Ledvina categorized HBPs according to the number of interdomain connections they contain: Group I have three interdomain connections and Group II have two connections (Quiocho & Ledvina,

1996). Recently, a third group of HBPs was identified; proteins in this group have domains joined by an single α-helix (Borths et al., 2002; Karpowich et al., 2003; Eakanunkul et al., 2005; Ho et al., 2007).

The HBP serves the same function in both Gram positive and negative bacteria (Clarke et al., 2001; Koster, 2001; Perkins-Balding et al., 2004; Debarbieux & Wandersman, 2004). Once loaded, the HBP delivers its Hm to the membrane permease, which is typically composed of hydrophobic α-helical domains (Koster, 2001; Higgins & Linton, 2004; Perkins-Balding et al., 2004; Linton & Higgins, 2007). The permease recognizes the heme-loaded HBP and provides a pathway for the Hm to cross the membrane. Transport is powered by the hydrolysis of ATP via the ATPase. This protein generally consists of two ATP binding domains. Heme ABC ATPases have relatively high sequence identity, and ATPases in general are thought to be the most highly conserved proteins in ABC transporters as compared to the binding and permease proteins (Perkins-Balding et al., 2004; Linton & Higgins, 2007).

3.4.2 Gram Negative Bacteria Heme ABC Systems. The heme ABC systems of Gram

negative bacteria are found within the cytoplasmic membrane (Clarke et al., 2001; Koster, 2001; Stojiljkovic & Perkins-Balding, 2002). Once Hm enters the periplasm, it is bound by the HBP of the ABC system, which in most Gram negative bacteria is soluble (Koster, 2001). The HBP then shuttles Hm to the membrane permease where it enters the cell.

The permeability limit of the Gram negative outer membrane is about 600 Da and

generally prevents hemophore passage through porins (Braun & Killmann, 1999). Thus, specific pumps are required to introduce Hm into the periplasmic space. To bring substrate into the periplasm, the bacteria rely on an outer membrane receptor and the TonB-ExbBD system (TonB) (Braun & Killmann, 1999; Ratledge & Dover, 2000; Koster, 2001; Clarke et al., 2001;

Stojiljkovic & Perkins-Balding, 2002; Wandersman & Delepelaire, 2004; Cescau et al., 2007; Miethke & Marahiel, 2007). The TonB protein is located in the periplasm and is anchored to the cytoplasmic membrane. It interacts across the periplasmic membrane with the outer membrane Hm receptor. The ExbB and ExbD proteins are also located in the cytoplasmic membrane. The TonB system uses ATP to mediate the transfer of Hm from the outer membrane to the periplasm. 3.4.3 Gram Positive Bacteria Heme ABC Systems. Gram positive bacteria are composed of a thick outer cell wall consisting mainly of peptidoglycan. Beneath the cell wall lies the cell membrane. The heme ABC systems for Gram positive bacteria are located in the cell membrane (Schmitt, 1997; Drazek et al., 2000). The HBP is attached to the cell membrane by a lipoprotein tail. Lipoprotein anchoring in Gram positive bacteria is thought to prevent the escape of cell envelope proteins (Sutcliffe & Russell, 1995; Sutcliffe & Harrington, 2002). Sutcliffe and Harrington describe the general anchoring process as being initiated by the secretion of a

prolipoprotein which contains a signal sequence at its N-terminal (Sutcliffe & Harrington, 2002). This signal sequence is followed by a lipobox motif which contains a conserved cysteine.

Prolipoprotein diacylglycerol transferase catalyzes the formation of a thioether linkage between the cysteine and a diglyceride. Lipoprotein signal peptidase II then removes the signal sequence. 3.5 Regulation of Transcription of Heme Acquisition Genes. Generally, expression of Hm and iron uptake systems in Gram negative bacteria (and some Gram positive bacteria) is negatively regulated by the Fur (ferric uptake regulation) protein (Ratledge & Dover, 2000; Clarke et al., 2001; Hantke, 2001; Brickman et al., 2004; Payne et al., 2006; Lee & Helmann, 2007; Wyckoff et al., 2007; Vasil, 2007). For example, in E. coli, the Fur protein forms a complex with Fe(II) (Earhart, 2004). This complex then binds to specific DNA sequences, which are generally located within the promoter region (de Lorenzo et al., 2004). Transcription

of iron uptake genes is repressed by blocking the attachment of RNA polymerase. In the Gram positive C. diphtheriae, the analogous protein DtxR (diphtheria toxin repressor) has been found (Braun, 1997; Ratledge & Dover, 2000; Hantke, 2001; Pennella & Giedroc, 2005; Bhattacharya et al., 2007; D'Aquino et al., 2007). DxtR-like proteins have also been located in other Gram positive bacteria. The DxtR and Fur families of proteins do not share homology, but they are structurally similar and perform similar duties in Gram positive and negative bacteria,

respectively.