Siderophore Synthesis Essay

Abstract

Iron serves as a signal in Pseudomonas aeruginosa biofilm development. We examined the influence of mutations in known and putative iron acquisition-signaling genes on biofilm morphology. In iron-sufficient medium, mutants that cannot obtain iron through the high-affinity pyoverdine iron acquisition system form thin biofilms similar to those formed by the parent under low iron conditions. If an iron source for a different iron acquisition system is provided to a pyoverdine mutant, normal biofilm development occurs. This enabled us to identify iron uptake gene clusters that likely serve in transport of ferric citrate and ferrioxamine. We suggest that the functional iron signal for P. aeruginosa biofilm development is active transport of chelated iron or the level of internal iron. If the signal is internal iron levels, then a factor likely to be involved in iron signaling is the cytoplasmic ferric uptake regulator protein, Fur, which controls expression of iron-responsive genes. In support of a Fur involvement, we found that with low iron a Fur mutant was able to organize into more mature biofilms than was the parent. The two known Fur-controlled small regulatory RNAs (PrrF1 and F2) do not appear to mediate iron control of biofilm development. This information establishes a mechanistic basis for iron control of P. aeruginosa biofilm formation.

Biofilms consist of groups of bacteria attached to surfaces and encased in a hydrated polymeric matrix. Pseudomonas aeruginosa biofilms cause persistent infections in individuals with underlying health problems. For example, most people with the genetic disease cystic fibrosis are plagued by chronic P. aeruginosa biofilm infections of their lungs (reviewed in refs. 1-3). Iron starvation can prevent bacterial growth. Recent work shows that with sufficient iron for growth the levels of this metal serve as a signal for biofilm development (4, 5). By sequestering iron, subgrowth inhibitory concentrations of the mammalian iron chelator lactoferrin block the ability of P. aeruginosa biofilms to mature from thin layers of cells attached to a surface into large multicellular biofilm structures (4). The influence of lactoferrin on biofilm development is thought to be related to the fact that at low iron concentrations P. aeruginosa exhibits incessant twitching motility on surfaces, and cells do not form sessile structures (4, 5). The mechanism for iron control of twitching motility is unknown, as is the mechanism of iron signaling in biofilm development.

Iron is essential for, yet toxic to, bacteria. For most pathogens, including P. aeruginosa, there is intense competition for iron with the host (6-8). P. aeruginosa has multiple systems to sense and sequester iron in its environment, and it is able to regulate cellular iron acquisition and storage through an assortment of positive and negative regulatory factors (reviewed in refs. 6, 9, and 10). The two best-studied P. aeruginosa iron acquisition systems are the high-affinity pyoverdine system and the lower-affinity pyochelin system. Pyoverdine and pyochelin bind extracellular iron (Fe3+), which is then transported into the cell together with these siderophores. Pyoverdine synthesis and secretion are regulated by means of the extracytoplasmic function (ECF) σ factor PvdS. Expression of PvdS is regulated by iron and the ferric uptake regulator (Fur). In this acquisition system, pyoverdine binds to an outer membrane-associated receptor, which results in transmission of a signal to a membrane-spanning anti-σ factor that governs the activity of PvdS, thereby controlling expression of pyoverdine synthesis. The signal also regulates expression of other genes, including extracellular virulence factors such as exotoxin A (9, 11, 12). There are at least 10 other gene clusters in the P. aeruginosa genome that may encode iron-responsive ECF σ factor-regulated systems (12). For example, there is one gene cluster encoding proteins with extensive sequence similarity to the FecIR, ferric citrate system in Escherichia coli (12, 13).

As in many other bacteria, P. aeruginosa has a Fur protein that functions as a global regulator of iron-responsive genes (14, 15). Fur has both negative and positive regulatory effects on gene expression. It represses gene expression by direct binding to the operators of iron starvation-inducible genes. It activates gene expression indirectly through control of a pair of small regulatory RNAs (sRNAs), PrrF1 and PrrF2 (16). In P. aeruginosa, fur appears to be an essential gene, and the only fur mutants available either produce reduced levels of wild-type Fur or have missense mutations that exhibit high reversion rates (17, 18).

We sought to better understand the iron-signaling cascade critical for normal biofilm development. We asked whether any of the known or putative iron-uptake and iron-starvation ECF σ factors are involved in regulating biofilm development. Our data indicate that any iron-uptake system that can provide sufficient levels of internal iron to P. aeruginosa can function in the iron-signaling pathway. This finding suggests that a critical level of intracellular iron serves as the signal for biofilm development. Our data also indicate that intracellular iron signaling is mediated by Fur but not by means of its regulation of PrrF1 and PrrF2.

Materials and Methods

Bacterial Strains, Plasmids, and Culture Conditions. We used P. aeruginosa PAO1 as the wild-type strain. Strain PAO1 and most of the P. aeruginosa transposon insertion mutants used were from the Comprehensive P. aeruginosa transposon mutant library at the University of Washington Genome Center (19). We used the following mutants with transposons in iron starvation ECF σ-factor homologs: PTL4103 (PA0149), PTL52421 (PA0472), PTL9556 (PA0675), PTL16978 (PA1300), PTL37473 (PA1363), P TL16034 (PA1912), P TL18118 (PA 2050), PTL51829 (PA3410), PTL7851 (PA3899), and PTL49853 (PA4896). We also used mutants with sequence similarity to various iron receptors: PTL40529 and PTL14626 (both PA0151); PTL20210 (PA0470) with similarity to a hydroxamate type receptor; PTL45373 (PA0674), a ferripyoverdine receptor homolog; PTL18189 (hxuC), a heme receptor homolog; PTL5195 (PA1365), a ferribactin receptor homolog; PTL4009 (PA1910); PTL11509 (PA2466), a ferrioxamine receptor homolog; PTL9628 (hasR), a heme uptake homolog; and PTL14263 (fecA). The pyochelin mutant (pchA) was PTL5032. All of the mutants contained either a ISlacZ/hah or an ISphoA/hah transposon insertion. Further information on these mutants is at www.genome.washington.edu/UWGC/Pseudomonas/index.cfm. The locations of all of the mutations were confirmed by PCR with primers flanking the insertion sites. We also used PAO1ΔpvdS (20); PAO1ΔpvdA (14); PAO1ΔfpvA (11), a strain with a fur point mutation; PAO1furC6Tc (17, 18), a PAO1ΔprrF1-F2 mutant (16); PAO1ΔpvdD ΔpchEF (provided by Urs Ochsner, Replidyne Inc., Louisville, CO); and several strains with mutations generated as described below.

For growth curve and flow cell biofilm experiments we used 1% Bacto Tryptic Soy Broth (TSB) (Becton Dickinson). Incubation was at room temperature (23-25°C). Where indicated, 20 μg/ml human lactoferrin (Sigma) was added to the medium. Also as indicated, 50 or 100 μM ferric chloride, 1 μM ferric dicitrate, 1.5 μM desferrioxamine mesylate (Sigma), or pyoverdine-conditioned medium was added. Pyoverdine-conditioned medium was prepared by growing P. aeruginosa PAO1 in Kings B medium (21) for 12 h at 37°C. The cells were removed by centrifugation and filtration through a 0.2-μm filter. Pyoverdine-conditioned medium was added at 1% as indicated. As a control for the pyoverdine experiments, we used medium conditioned by the PvdA mutant PAO1ΔpvdA. Escherichia coli was used for recombinant manipulations and was grown in Luria-Bertani (LB) broth. We used Pseudomonas Isolation Agar (Becton Dickinson) for selection of transconjugants. Antibiotics were used at the following concentrations: 100 μg/ml gentamicin (Gm) for E. coli and P. aeruginosa,10 μg/ml tetracycline (Tc) for E. coli, and 100 μg/ml Tc for P. aeruginosa.

Construction of Plasmids and P. aeruginosa Mutants. Although most of the P. aeruginosa mutants were from a preexisting library, some were constructed as described here. For construction of the PA2387 mutant (EB101), a 1.4-kb HindIII-NotI DNA fragment upstream of PA2387 (from bp 2638897 to bp 2640299 on the P. aeruginosa chromosome) and a 1.5-kb NotI-XbaI DNA fragment downstream of PA2387 (from bp 2640893 to bp 2674792 on the P. aeruginosa chromosome) were amplified and cloned into a HindIII-XbaI-digested pEX18Tc gene replacement vector (22). The resulting plasmid was digested with NotI and ligated with a NotI-digested aacC1 cassette to create pEB2. This plasmid was mobilized from E. coli SM10 (23) into P. aeruginosa PAO1 by conjugation. Selection for GmR colonies followed by screening for TcS colonies yielded a PA2387 mutant, P. aeruginosa EB101. The mutation was confirmed by PCR analysis. The PA2467-2468 mutant (EB102) was constructed in a similar fashion. A 1.4-kb HindIII-NotI DNA fragment corresponding to a region upstream of PA2467 (from bp 2783773 to bp 2785221 on the P. aeruginosa chromosome) and a NotI-XbaI 1.5-kb fragment downstream of PA2468 (from bp 2786704 to bp 2788240 on the P. aeruginosa chromosome) were amplified and cloned into HindIII-XbaI-digested pEX18Tc. The resulting plasmid was then digested with NotI and ligated with a NotI-digested aacC1 cassette to create pEB3. A transconjugant was isolated, and the mutation was confirmed as described above.

We constructed the following double mutants using a method described in ref. 24: EB104 (PAO1 ΔpvdA,ISlacZ/hah PA3901), EB107 (PAO1 ΔpvdA,ISlacZ/hah pchA), EB110 (PAO1 ΔpvdA, ISlacZ/hah PA0470), and EB111 (PAO1 ΔpvdA, ISlacZ/hah PA2466). In all cases we transformed PAO1ΔpvdA with genomic DNA (40-80 μg) from PTL14263 to construct EB104, PTL5032 to construct strain EB107, PTL20210 to construct strain EB110, or PTL11509 to construct strain EB111. After transformation, colonies were selected on LB agar plates containing Tc (100 μg/ml). The mutation in each strain was confirmed by PCR.

The triple mutant EB112 (PAO1ΔpvdA, 63-bp insertion in PA0470, ISlacZ/hah PA2466) was constructed by using the cre-recombinase system (25). Strain EB110 was mated with E. coli carrying pCre1. Transconjugants were selected on Pseudomonas isolation agar. A TcS strain was isolated, and the 63-bp insertion was confirmed by PCR. This strain was transformed with PTL11509 genomic DNA, a transformant was selected, and all of the mutations were confirmed by PCR.

To construct the pvdS-gfp fusion strain, a 250-bp EcoRI-HindIII fragment containing the pvdS promoter was amplified (from bp 2721925 to bp 2722175) and cloned into an EcoRI- HindIII-digested promoterless gfp pPROBE AT (26). The plasmid was transferred to PAO1ΔpvdD ΔpchEF by conjugation. The resulting strain, EB125, contains the pvdS-gfp fusion as a single chromosomal copy.

For complementation of the pvdA mutation, a 2-kb fragment containing pvdA was amplified (from bp 2638500 to bp 2640501) and cloned in EcoRI-digested pJN105 (27). The resulting plasmid pEB4 was used to transform PAO1ΔpvdA. For complementation of the pvdS mutation we used pPVD27 (28).

Biofilm Experiments. We used flow cells to follow biofilm growth on a glass surface (24). The flow cells were inoculated with a 1:50 dilution (in 1% TSB) of a P. aeruginosa stationary phase culture, and flow was initiated after 1 h. To image biofilms, we used confocal scanning laser microscopy (CSLM). The CSLM was a Radiance 2100 system (Bio-Rad) with a Nikon Eclipse E600 microscope. Generally, we imaged GFP in P. aeruginosa containing pMRP9-1, which has a constitutively expressed gfp (29). Where indicated, we counterstained the biofilm with propidium iodide (4 μM) and imaged the propidium iodide fluorescence by CSLM (30). The image acquisition software was lasersharp 2000 (Bio-Rad). Images were processed with confocal assistant or volocity (Improvision, Lexington, MA) software.

Twitching Motility Assays. For twitching motility assays we used Petri plates with 1% TSB plus 1% Noble agar (Becton Dickinson) with or without 2 mM desferrioxamine to chelate iron as indicated. Plates were dried overnight at room temperature, and cells from a colony grown overnight on a LB-agar plate were point inoculated at the bottom of the agar plate. After 3 days, the twitching motility distance along the plastic-agar interface (at the bottom of the agar plate) was measured.

Results

Biofilm Development in Pyoverdine and Pyochelin Synthesis Mutants. We first followed biofilm development in P. aeruginosa mutants incapable of synthesizing the two well known siderophores pyoverdine and pyochelin, PAO1ΔpvdA and PAO1pchA::TcR, respectively. Planktonic growth in 1% TSB and twitching motility (data not shown) of these mutants were indistinguishable from the parent. Biofilm formation of the pyochelin mutant under a flow of 1% TSB is similar to that of the parent. Both strains form mushroom-like structures on the glass surface. The pyoverdine mutant, however, forms a thin uniform layer of growth on the glass surface that is similar to the biofilms formed by the parent and pyochelin mutant in the presence of lactoferrin (Fig. 1). PvdA is required for pyoverdine synthesis, and PvdS codes for the σ factor required to activate pyoverdine synthesis and to regulate expression of other genes. A pvdS mutant, PAOΔpvdS, shows a biofilm phenotype similar to that of the pvdA mutant (Fig. 1). Complementation of the pvdA and pvdS mutations restores the normal biofilm architecture (Fig. 1). Furthermore, addition of pyoverdine-conditioned medium (see Materials and Methods) allows biofilms of the PvdA mutant to form normally (Fig. 1).

Fig. 1.

Biofilm formation of P. aeruginosa pyoverdine and pyochelin mutants. The parent without (A) and with (B) lactoferrin (20 μg/ml), the pyochelin mutant PAO1pchA::TcR without (C) and with (D) lactoferrin (20 μg/ml), the pyoverdine synthesis mutant PAO1ΔpvdA (E), the pyoverdine ECF σ-factor PvdS mutant PAOΔpvdS without lactoferrin (F), the pyoverdine mutant carrying the pvdA expression vector pEB4 without lactoferrin (G), and the pyoverdine mutant grown in pyoverdine-conditioned medium without lactoferrin (H). The P. aeruginosa cells contained the gfp plasmid pMRP9-1. Images are from 6-day biofilms (the squares are 61 μm on a side).

Biofilm Formation in Flow Cells Requires Active Iron Transport. Compared to pyoverdine, pyochelin has a low affinity for iron (9). To ask whether the pyoverdine mutant can acquire iron for signaling biofilm development through pyochelin in the presence of sufficiently high environmental iron concentrations, we supplemented the medium with 50 μM FeCl3. In this iron-rich medium the pyoverdine mutant produces biofilms that are similar to the parent. A pvdA,pchA double mutant, EB107, forms thin, flat biofilms in high-iron medium, even at 100 μM ferric chloride (Fig. 2). We obtained similar results with a pvdD,pchEF double mutant (data not shown). Addition of desferrioxamine or ferric dicitrate, both of which P. aeruginosa can use as an iron source (31, 32), restores biofilm formation by the double mutant (Fig. 2). These experiments suggest that an iron uptake system is required for normal biofilm development on glass and that passive diffusion of iron does not provide sufficient intracellular iron for biofilm development. To test the hypothesis that P. aeruginosa biofilms cannot obtain sufficient iron for signaling by passive diffusion even at high external iron concentrations, we examined the expression of an iron-repressed, Fur-regulated promoter (pvdS)-driven gfp in our pyoverdine, pyochelin double mutant (EB125). With or without added iron (100 μM FeCl3), biofilms are flat (<15 μm), and the cells express GFP (Fig. 3). In the presence of added iron, the cells on the biofilm surface express less GFP than those at the base of the biofilm (Fig. 3). This finding suggests that there is sufficient iron for Fur to serve as a repressor in cells near the biofilm surface, but without an iron uptake system passive diffusion does not provide sufficient iron for Fur repression throughout the biofilm. When the double mutant is grown in the presence of desferrioxamine, which can be taken up by the ferrioxamine transport system, mushroom-like structures form and the cells express low levels of GFP (Fig. 3). These data are consistent with the hypothesis that an active iron transport system is needed for normal biofilm development.

Fig. 2.

Active iron transport is required for normal biofilm development in flow cells. (A) Biofilm of the pvdA mutant PAO1ΔpvdA grown with added ferric chloride (50 μM). Biofilm of a pyoverdine, pyochelin double mutant (EB107) grown with 100 μM ferric chloride (B) or 1.5 μM desferrioxamine (C) (similar results were obtained when 1 μM ferric dicitrate was added; data not shown). All strains contained the gfp plasmid pMRP9-1. Images represent 6-day biofilms (the squares are 30 μm on a side).

Fig. 3.

Expression of pvdS-gfp in biofilms of a pyoverdine, pyochelin double mutant (EB125) in the presence of FeCl3 or desferrioxamine. (A) Biofilms grown without FeCl3.(B) Biofilms grown with 100 μM FeCl3.(C) Biofilms grown with 1.5 μM desferrioxamine. Images represent 6-day biofilms counterstained with propidium iodide. (A-C Upper) A reconstructed side view of the combined red and green channels. Red shows the biofilm, and green shows regions where cells have expressed GFP. (A-C Lower)A side view of GFP expression alone. (Scale bar in Lower, 15 μm; all of the side images are the same magnification.)

Do Mutations in Other Iron-Responsive ECF σ-Factor Systems Influence Biofilm Formation? Visca et al. (12) identified 19 ORFs encoding putative ECF σ-factors in the P. aeruginosa genome. Thirteen of these show substantial sequence similarity with iron starvation σ factors, and they contain putative Fur binding sites in their promoter region. Several are in clusters that include genes predicted to encode outer-membrane receptors involved in iron transport. We wanted to determine whether any of these putative iron acquisition signaling systems is involved in biofilm development; therefore, we studied mutants with defects in the putative iron-responsive ECF σ-factor genes and the putative FecA iron receptor homologs. Mutants with insertions in the following ECF σ-factor genes were examined: PA0149, PA0472, PA0675, PA1300, PA1363, PA1912, PA2050, PA2387, PA2468, PA3410, PA3899, and PA4896. Iron receptor mutants with insertions in PA0151, PA0470, PA0674, PA1302, PA1910, PA1365, PA2398, PA2466, PA3408, and PA3901 were also examined. None of the mutations has an appreciable affect on planktonic growth or twitching motility (data not shown). With a single exception, biofilm formation of the mutants is indistinguishable from the parent in the presence (thin, flat biofilms) or absence (large, mushroom-like structures) of lactoferrin. The single exception is the PA2398 mutant, PAO1fpvA::GmR. This fpvA mutant forms thin, flat biofilms in the absence of lactoferrin that are similar in appearance to those formed by pvd mutants in the absence of lactoferrin (data not shown). FpvA is known to function in the pyoverdine signaling and uptake cascade (33).

Alternate Sources of Iron Can Signal Biofilm Development in a Pyoverdine Synthesis Mutant. Without sufficiently high levels of environmental iron, pyoverdine mutants form thin, flat biofilms (Fig. 1), and we believe that cytoplasmic iron levels or transport of chelated iron cues biofilm development. However, we know that P. aeruginosa can use a variety of chelated forms of environmental iron and that it can acquire iron chelated by other organisms (9). For example, it can use iron chelated with citrate or with desferrioxamine (31, 32, 34). If iron signaling in biofilm formation is mediated by a cytoplasmic receptor or active transport of chelated iron, then provision of ferric dicitrate or desferrioxamine should allow the pvdA mutant to form biofilms with the wild-type mushroom-like structures. In fact, biofilms of the pvdA mutant grown in the presence of 1 μM ferric dicitrate show the mushroom-like appearance of the parent strain (Fig. 4).

Fig. 4.

Ferric citrate and ferrioxamine mediate development of mushroom-like structures in the pyoverdine synthesis mutant PAO1ΔpvdA. The pvdA mutant biofilm in medium supplemented with 1 μM ferric dicitrate (A) or 1.5 μM desferrioxamine (B). (C) A biofilm of the pvdA, PA3901 double mutant EB104 in medium containing 1 μM ferric dicitrate. (D) A biofilm of EB112 (the pyoverdine, PA2466, and PA0470 triple mutant) in medium with 1.5 μM desferrioxamine. All strains contained pMRP9-1. Images represent 6-day biofilms (the squares are 62 μm on a side).

The P. aeruginosa PA3899-3901 gene cluster codes for polypeptides showing extensive sequence identity with the E. coli ferric dicitrate uptake polypeptides, the Fec polypeptides (35). Thus, we constructed P. aeruginosa EB104, which contains an insertion in PA3901 and a deletion in pvdA. This mutant shows no appreciable differences in growth and twitching motility compared with the parent; however, it forms thin, flat biofilms even in the presence of ferric dicitrate (Fig. 4). Similar to the results obtained with ferric dicitrate as an alternative iron scavenger, addition of desferrioxamine (1.5 μM) also restores mushroom formation to the pvdA mutant. In the P. aeruginosa genome there are two gene clusters (PA0470-0472 and PA2466-2468) that show extensive sequence identity with ferrioxamine uptake systems from other bacteria (15). We constructed two double mutants that contain a deletion in pvdA and an insertion in PA0470 or PA2466 (EB110 and EB111, respectively). Both mutants form mushroom-like structures in the presence of desferrioxamine (data not shown). A triple mutant (EB112) that contains deletions in pvdA and PA2466 and an insertion in PA0470 forms a sparse biofilm (Fig. 4). This triple mutant exhibits normal twitching motility, but its growth in broth containing desferrioxamine is poor (data not shown). These data are consistent with the conclusion that with sufficient levels of cytoplasmic iron P. aeruginosa biofilms form mushroom-like structures. These experiments also provide evidence suggesting that PA3899-3901 are involved in ferric dicitrate uptake and that the PA0470 and PA2466 gene clusters are involved in ferrioxamine uptake. Further work is required to critically establish these gene clusters as ferric dicitrate and desferrioxamine uptake systems.

Intracellular Iron Signaling Is Mediated by Fur. The experiments described above are consistent with the hypothesis that biofilm development is regulated by intracellular iron. The major intracellular iron regulator in Proteobacteria including P. aeruginosa is Fur (6, 15). The P. aeruginosa Fur appears to be an essential gene; however, Fur missense mutants have been isolated (17, 18). Fur is an iron-dependent transcription repressor. Thus, one might predict that a P. aeruginosa Fur mutant could build structured biofilms even under lactoferrin-induced iron limitation. Accordingly, we followed biofilm development of a Fur mutant in the presence and absence of lactoferrin. Without lactoferrin the mutant forms mushroom structures that resembled parent structures without lactoferrin (Fig. 5). In the presence of lactoferrin, the mutant forms structured biofilms whereas the parent forms the expected flat, thin biofilms (Fig. 5). These observations suggest that Fur controls genes that are crucial in biofilm development.

Fig. 5.

Biofilm formation by Fur and PrrF mutants. (Top) Biofilms of the parent PAO1. (Middle) Biofilms of the Fur mutant PAO1furC6Tc. (Bottom) Biofilms of the PAO1ΔprrF1-F2 double mutant. Where indicated, biofilms were grown in the presence of 20 μg/ml lactoferrin. Strain PAO1 and PAO1ΔprrF1-F2 contained the gfp plasmid pMRP9-1. The Fur mutant was stained with propidium iodide (we could not introduce pMRP9-1 into the strain). Images in Left are of 6-day biofilms, and the squares are 60 μm on a side. Images in Right are also images of 6-day biofilms, but the squares are 30 μm on a side.

Wilderman et al. (16) recently identified two Fur-repressed sRNA molecules in P. aeruginosa. These sRNAs (PrrF1 and PrrF2) are >95% identical to each other, and expression of both is repressed by iron. However, each can also be independently regulated by other factors (e.g., heme). Deletion of both sRNAs is required to affect the iron-dependent regulation of an array of genes, including those involved in resistance to oxidative stress, iron storage, and intermediary metabolism. Unlike the Fur mutant, our PrrF1F2 double mutant formed biofilms similar to the parent mushroom-like structures in the absence of lactoferrin and thin, flat biofilms in the presence of lactoferrin (Fig. 5). Thus, we conclude that these sRNAs are not required for biofilm formation under the conditions used in this study.

Discussion

Singh et al. (4) discovered that the mammalian iron chelator lactoferrin restricts the development of P. aeruginosa biofilms. In the presence of lactoferrin, P. aeruginosa forms a thin layer of cells on glass surfaces and the individual cells exhibit incessant twitching motility. In the absence of lactoferrin, P. aeruginosa constructs thicker biofilms consisting of cells clustered in mushroom-like structures. The lactoferrin effect was correlated to its activity as an iron chelator. These experiments indicated that iron serves as an environmental signal for P. aeruginosa biofilm development. To better understand the role of iron in P. aeruginosa biofilm formation, we studied a variety of mutants with defects in known or suspected iron-uptake and regulatory functions. In the absence of a functional iron uptake system, P. aeruginosa forms thin, flat biofilms in the absence of lactoferrin (Fig. 1). We show that P. aeruginosa can acquire iron and support normal biofilm formation by using the endogenous siderophores pyoverdine (at relatively low external iron concentrations) or pyochelin (at higher iron levels). It can also use specific iron-responsive ECF σ-factor systems to acquire ferric citrate or ferrioxamine for biofilm formation (Figs. 1, 2, 3, 4). It cannot acquire sufficient amounts of iron for normal biofilm development by passive diffusion in the flow-cell system (Fig. 3). All of our data on biofilm formation by iron acquisition mutants are consistent with the conclusion that biofilm formation in flow cells requires active iron transport and sufficient intracellular iron serves as a cue for development of mushroom-like structures. Although the pyoverdine uptake system has a dual function in that it also serves to regulate expression of a number of virulence genes directly (11, 36), this does not appear to be its role in iron regulation of biofilm formation.

Strains with mutations in the other predicted iron uptake systems formed normal biofilms. This finding is inconsistent with a recent report that a specific virulence mutant had a defect in a putative iron receptor (PA0151) and had a defect in biofilm formation (37). We tested two PA0151 mutants, and both formed normal biofilms in the absence of lactoferrin. This discrepancy could result from the differences in experimental protocols or strain variation. Our results show that, under the conditions we used and with derivatives of strain PAO1, normal biofilms form as long as there is a functional pyoverdine system and there is a sufficient level of environmental iron for the pyoverdine system to function.

As discussed above, a mutant strain that could not produce the high-affinity siderophore pyoverdine or the low-affinity siderophore pyochelin is unable to form mushroom-like structures even in the presence of high levels of inorganic iron, but it can form these structures when given ferric dicitrate or desferrioxamine (Fig. 2). We know that P. aeruginosa can use ferric citrate and other chelated forms of iron (9, 31, 33), but there is no experimental evidence linking specific genes to ferric dicitrate utilization. Among the various mutants with specific defects in putative iron-responsive ECF σ-factor systems that we tested, there is one with a mutation in the PA3899-3901 gene cluster. The polypeptides coded by genes in this cluster show >50% identity to the E. coli ferric uptake gene products FecA, FecI, and FecR (35). This E. coli system is responsible for ferric dicitrate uptake. We thus constructed a mutant incapable of producing pyoverdine and this putative ferric citrate system. This mutant produced thin, flat biofilms even when ferric dicitrate was provided (Fig. 4). This finding suggests that the PA3899-3901 cluster codes for a ferric dicitrate uptake system. We also have similar data suggesting that two gene clusters PA2466-2468 and PA0470-0472 facilitate ferrioxamine uptake (Fig. 4). Further characterization of these gene clusters is required to verify their role in iron transport. We are currently employing this screening approach to correlate specific iron-responsive ECF σ-factor systems with the ability to use different chelated forms of iron for biofilm development.

If, as we conclude, cytoplasmic iron functions as the signal for development of mushroom-like structures, a next question is what is the intracellular iron response regulator? A logical candidate is Fur, the major iron-responsive transcriptional regulator in P. aeruginosa (15). If Fur controls a gene or genes required for biofilm development into mushroom-like structures, one might predict that a Fur mutant could form such clusters in the presence of lactoferrin where the parent forms thin, flat biofilms. We tested a strain with a point mutation in this essential gene. This mutation results in low Fur activity (17). The influence of lactoferrin on the biofilm development of this mutant was not as severe as the influence of lactoferrin on the parent. Whereas the parent formed flat, thin biofilms in the presence of lactoferrin, the mutant formed large clusters of cells reminiscent of, but not identical to, the parent in the absence of lactoferrin (Fig. 5). Fur appears to mediate iron signaling in biofilm development through its regulation of multiple iron acquisition systems and their regulatory genes (e.g., ECF σ factors). The fact that Fur mutants produce high amounts of pyoverdine (17) may account in part for the biofilm architecture of our Fur mutant. We do not know what other Fur-regulated factors might be involved in biofilm formation. Nevertheless, we ruled out a contribution of the Fur-repressed sRNAs in biofilm growth (Fig. 5).

Biofilm development is thought to occur through a series of discrete steps. The first step involves attachment of cells to a surface. The second step is microcolony formation. A third step involves maturation of microcolonies into mushroom-like structures. Finally, there is a detachment step. Previous work suggests that iron sensing might serve as a checkpoint in the first two steps. O'Toole and Kolter (38) reported that the defect in certain attachment mutants can be overcome if sufficient iron is added to the medium. Singh et al. (4) showed that low iron stimulates twitching motility and blocks the formation of microcolonies on glass surfaces. Our data suggest that there may be another iron checkpoint for development of microcolonies that is not related to twitching. In support of a third checkpoint, the pyoverdine mutants (PvdA, FpvA, and PvdS) did not show a twitching motility phenotype. Furthermore, time-lapse microscopy of the mutants developing as biofilms did not show the incessant twitching described by Singh et al. (data not shown). The Fur mutant appears to bypass all three hypothetical checkpoints and form mushroom-like structures even in the presence of lactoferrin.

Previous studies showed that the formation of mushroom-like structures by P. aeruginosa biofilms depended on the carbon and energy source provided in the growth medium (39, 40). For biofilms grown in minimal medium, glucose supported formation of mushroom-like structures, but citrate did not. This finding may suggest that the iron-regulatory pathway and the carbon source-regulatory pathway might converge at a point downstream of Fur involvement.

It is not surprising that multiple iron uptake systems play a role in biofilm formation. It is likely that the different environments where P. aeruginosa resides dictate which forms of iron might be available. For example, ferric citrate or ferrioxamine are not likely to be available iron sources in a mammalian host. Finally, it must be said that no one yet understands the significance of the formation of mushroom-like structures on glass surfaces to biofilm formation in natural environments. Nevertheless, understanding the pathways leading to formation of these structures informs us about regulatory networks involved in biofilm development. It seems clear that iron plays a critical role in biofilm formation. It may be that there are several iron-regulated steps in biofilm formation. We show that at least one of these depends on the ability of cells to import sufficient levels of iron for Fur activity.

Acknowledgments

We thank Michael Jacobs, Colin Manoil, and Maynard Olson for providing us with the P. aeruginosa mutants from the University of Washington Genome Center P. aeruginosa PAO1 transposon mutant library. We thank Pradeep Singh for his advice and comments. This work was supported by a grant from the W. M. Keck Foundation (to E.P.G.), National Institute of General Medical Sciences Grant GM59026 (to E.P.G.), and National Institute of Allergy and Infectious Diseases Grant AI15940 (to M.L.V.). E.B. was partially funded by the Fulbright program.

Footnotes

  • ↵‡ To whom correspondence should be addressed. E-mail: epgreen{at}u.washington.edu.

  • Author contributions: E.B. and E.P.G. designed research; E.B. performed research; E.B., M.L.V., and E.P.G. analyzed data; E.B., M.L.V., and E.P.G. wrote the paper; and M.L.V. contributed new reagents/analytic tools.

  • Abbreviations: ECF, extracytoplasmic function; Tc, tetracycline; Fur, ferric uptake regulator; TSB, Bacto Tryptic Soy Broth; sRNA, small regulatory RNA.

  • Copyright © 2005, The National Academy of Sciences

References

Siderophores can be divided into three main classes depending on the chemical nature of the moieties donating the oxygen ligands for Fe(III) coordination, which are either catecholates (sensu stricto, catecholates and phenolates; better termed as “aryl caps”), hydroxamates, or (α-hydroxy-)carboxylates. However, increasing information about new siderophores led to a more complex classification since many structures that integrate the chemical features of at least two classes into one molecule, resulting in “mixed-type” siderophores, are meanwhile known. Some representative structures of various siderophore types are shown in Fig. ​1.

Physicochemical Properties of Siderophores

Siderophores are designed to form tight and stable complexes with ferric iron. Generally, the hard Lewis acid Fe(III) is strongly solvated in aqueous solution, forming an octahedral Fe(H2O)63+ complex (73). Due to the gain of entropy, the siderophore donor atoms favorably replace the solvent water and surround Fe(III) in a hexacoordinated state that usually has an octahedral geometry as it is found in the aqueous ion. Provided that the siderophore contains six donor atoms, a 1:1 complex with Fe(III) is generally formed. If there are less than six donor atoms provided by the ligand, the vacancies may be occupied by alternative oxygen donors such as water molecules, or siderophore complexes with higher stoichiometry may be built up as in the cases of rhodoturolic acid that forms Fe2L3 complexes (46), pyochelin that forms both FeL and FeL2 complexes (319), or cepabactin that forms FeL3 complexes (168). Even the formation of mixed complexes was observed for cepabactin and pyochelin, forming 1:1:1 complexes with Fe(III) (168). The diverse formation of iron-siderophore complexes with higher stoichiometry is strongly dependent on ligand concentration and protonation (319). The bound Fe(III) is always found in a high-spin d5 electronic configuration in the siderophore complex. Although there is no ligand field stabilization energy provided by this configuration, the complex is also kinetically stable, since the oxygen donor atoms that are mainly used in siderophores for iron coordination represent hard Lewis bases that allow additional strong ionic interactions between metal and ligand. Thus, siderophores display an enormous affinity towards Fe(III). Fe(III)-siderophore complexes often show characteristic UV-visible and circular dichroism spectra, and as there are no spin-allowed d-d transitions of the metal center in the case of bound Fe(III), the spectra are caused by ligand-to-metal or ligand-to-ligand charge transfer (271). The borderline Lewis acid Fe(II), in contrast, prefers interaction with softer donor atoms such as nitrogen or sulfur, which are only partially employed for iron coordination in natural siderophores. Furthermore, the higher Fe(II) electron density is poorly compensated for in the oxygen-donor atom-dominated siderophore complexes, and the low ratio of charge to ionic radius of Fe(II) compared with that of Fe(III) might be additionally unfavorable to maintain the optimal complex geometry. Thus, the capability of siderophores of forming stable complexes with Fe(II) is rather low.

In the thermodynamics of iron-siderophore binding, formation constants (Kf values) are attractive primarily for comparisons of iron affinities of various siderophores. In general, the overall equilibria of metal-ligand stability constants are expressed by a standard convention as βmlh values for the reaction mM + lL + hH = MmLlHh, where M is metal, L is ligand, and H is proton(s). For the wide variety of siderophores known so far, the corresponding formation constants for iron binding to the fully deprotonated ligand (Kf ☰ β110) ranges over about 30 orders of magnitude. This enormous affinity range is representatively demonstrated by two E. coli siderophores: the mixed citrate-hydroxamate aerobactin as one of the weakest monohexadentatic iron chelators, with a log β110 of 22.5 (133), and the triscatecholate enterobactin as the strongest iron-chelating compound ever found, displaying a log β110 of 49 (198). However, since protonation of the donor atoms is a competitive reaction to metal chelation, the pKa values of the donor groups have to be considered in terms of effectiveness of iron complexation. Catecholate siderophores have pKa values from 6.5 to 8 for the dissociation of the first hydrogen and about 11.5 for the second hydrogen from the catecholic hydroxyl groups. Hydroxamates show pKa values from 8 to 9. The pKa values of carboxylates ranging from 3.5 to 5 make them efficient siderophores under lower-pH conditions at which catecholates and hydroxamates are still fully protonated. Thus, microbes living in acidic habitats, such as many fungi, tend to prefer carboxylate siderophores for iron mobilization, although they could not compete with stronger siderophores such as catecholates at physiological pH (74).

Because proton-independent Kf values do not reflect the real iron binding capacity of the siderophores under physiological conditions for which complete deprotonation is usually not achieved, a more convenient measure for comparing the true relative abilities of different siderophores to bind ferric iron is the pH-analogous pFe value, which gives the negative decadic logarithm of the free iron concentration if, according to a standard convention, the total Fe(III) concentration is 10−6 M and the total ligand concentration is 10−5 M. Since the pH of the medium strongly influences the chelation efficiency, pFe is a pH-dependent value. In this respect, only at above a pH of ∼5.0 is enterobactin significantly more efficient as an iron chelator than aerobactin (322). At a serum pH of 7.4, the free iron concentrations in the presence of enterobactin (pFe[pH 7.4] = 35.5) and aerobactin (pFe[pH 7.4] = 23.4) differ by more than 10 orders of magnitude (73).

Furthermore, the iron affinity of the siderophore determines the redox potential of the ferric iron within the complex. While the standard redox potential (E°) of the Fe(III)/Fe(II) couple in water is +0.77 V, iron chelation can change the E° of the ligand-Fe(III) complex [LFe(III)]/LFe(II) couple dramatically. It may increase for Fe(II) complexes such as Fe-phenanthroline or Fe-bipyridyl above 1 V, meaning that Fe(II) in the complex will not be available any more as a reducing agent, e.g., for the Fenton reaction. On the other hand, it may decrease below −0.4 V in the case of Fe(III) chelates such as ferrioxamine B and Fe-enterobactin, with E° values at pH 7.0 of −0.45 V and −0.75 V, respectively, meaning that the chelated Fe(III) is not accessible to reduction by biological reducing agents such as NAD(P)H or flavins (251). Generally, increasing formation constants of Fe(III) complexes result in decreasing redox potentials of the bound Fe(III). However, the actual redox potential, E, does not necessarily correspond with E°, since it depends furthermore on the ratio of the current LFe(III) and LFe(II) concentrations: E = E° + 0.059 log[LFe(III)]/log[LFe(II)]. Thus, if the concentration of LFe(II) is reduced, which can be achieved in biological systems by Fe(II) sequestration into apoproteins or porphyrins or by transport processes, E is significantly increased, and the reduction of complexed Fe(III) by usual biological reducing agents may become thermodynamically favorable (251). An increase in the Fe(III)-chelate redox potential may additionally occur by reducing the pH (in the periplasm or in vacuoles) or increasing the hydrophobicity (by membrane association) of the reaction environment.

Gene Regulation of Microbial Iron Homeostasis

For the utilization of siderophores, microorganisms have to tightly regulate enzymes and transport systems that allow concerted siderophore biosynthesis, secretion, siderophore-delivered iron uptake, and iron release. In bacteria, gene regulation of siderophore utilization and iron homeostasis in general is mediated mainly at the transcriptional level by the ferric uptake repressor Fur or the diphtheria toxin regulator DtxR (131). While Fur is the global iron regulator in many gram-negative (e.g., enteric bacteria) and low-GC-content gram-positive (e.g., Bacillus spp.) bacteria, DtxR fulfills a comparable role in gram-positive bacteria with a high GC content (streptomycetes, mycobacteria, and corynebacteria). In bacteria that regulate iron homeostasis by Fur, DtxR-like proteins generally regulate manganese transport. Although there are no obvious sequence similarities present, the structural aspects of both regulators are very similar in terms of domain organization and metal binding (253-255). The C-terminal domains involved in homodimer formation contain a structural binding site for Zn(II) and a regulatory binding site for Fe(II). Zn(II) binding was shown to mediate the dimerization of E. coli Fur, although the Zn(II) binding site seems not to be strictly conserved among all Fur orthologues (248). Fe(II) binds to the regulatory site as a corepressor and was shown to enable E. coli Fur to bind to its DNA recognition sites (Fur boxes) (14). The first structural analyses were performed with DtxR from Corynebacterium diphtheriae. An Mn(II)-DtxR structure in the presence of either sulfate or selenate allowed the identification of an anion binding site near metal binding site I, leading to the suggestion that phosphate might act as a “co-corepressor” of DtxR under physiological conditions (261). Crystallization of the DtxR homologue IdeR from Mycobacterium tuberculosis as well as Pseudomonas aeruginosa Fur in the presence of 10 mM Zn-acetate and 10 mM ZnSO4, respectively, led to structures of both regulators in the homodimeric state with the two metal binding sites per monomer fully occupied by Zn(II) (253, 254). Concomitantly, extended X-ray absorption spectroscopy analyses with the Fur protein in solution confirmed the postulated redox state of Fe(II) bound to the regulator, and it was suggested that Zn(II) binding site I observed in the Zn(II)-Fur structure might represent the regulatory Fe(II) binding site in vivo (253). The putative Fe(II) binding site is composed of five amino acid residues (two His residues, two Asp residues, and one Glu residue) and one water molecule, suggesting an octahedral ligand arrangement.

In addition to the global repression systems, various transcriptional regulators of a lower hierarchy are involved in the regulation of siderophore utilization in bacteria. Generally, they act as activators, which are functionalized by direct or indirect sensing of extra- or intracellularly present Fe-siderophores and can be grouped into different classes including (i) alternative sigma factors, (ii) two-component sensory transduction systems, (iii) AraC-type regulators, and (iv) further transcriptional regulator types.

In the first class, the E. coli extracytoplasmic function (ECF) sigma factor FecI regulates the fecABCDE ferric citrate transport genes by the indirect sensing of extracellular ferric dicitrate via its cognate outer membrane (OM) receptor FecA, the N-terminal periplasmic region of which transmits the siderophore binding signal to the C terminus of FecR, the membrane sensor factor located in the cytoplsamic membrane. FecR interacts via its cytoplasmic N-terminal domain with FecI and thus may function as both an FecI chaperone and an anti-sigma factor (88). Homologous systems of FecI-FecR-FecA are PupI-PupR-PupB and FpvI/PvdS-FpvR-FpvA, regulating pseudobactin (type of linear, pyoverdin-like siderophore) and pyoverdin utilization in Pseudomonas putida and P. aeruginosa (31). In these systems, PupR is the anti-sigma factor of PupI, and FpvR is the anti-sigma factor of both ECF sigma factors FpvI and PvdS. The FecI homologue PbrA of Pseudomonas fluorescens activates the transcription of several iron utilization genes encoding pseudobactin biosynthesis and Fe-pseudobactin receptors (65). Several pathogenic Bordetella spp. regulate heme utilization via homologous ECF sigma factor-dependent systems.

The two-component sensory transduction system PfeR-PfeS of P. aeruginosa induces the pfeA Fe-enterobactin receptor by sensing periplasmic Fe-enterobactin (71).

AraC-like transcriptional regulators represent fusion proteins of AraC-type DNA binding domains and various substrate binding domains that bind Fe-siderophores as coregulators. Such intracellular siderophore sensors are found among both pathogenic and nonpathogenic siderophore-utilizing bacteria and may have evolved in terms of fine-tuned siderophore pathway regulation by directly responding to the presence of the iron chelator either before secretion or after uptake as an iron-charged complex. The first described member of this class was the PchR regulator of P. aeruginosa, which induces the pyochelin biosynthesis genes pchDCBA and the Fe-pyochelin receptor ftpA and represses its own gene in the presence of Fe-pyochelin (214). The AlcR regulator of Bordetella pertussis and Bordetella bronchiseptica was found to induce the alcaligin biosynthesis genes alcABCDER and the Fe-alcaligin receptor fauA in response to alcaligin (36). Recently, the Btr (YbbB) regulator of Bacillus subtilis was identified as being another siderophore-binding AraC-type regulator. Btr binds to the promoter of the Fe-bacillibactin uptake operon feuABC ybbA in the absence of bacillibactin (formerly also termed “corynebactin”) but needs Fe-bacillibactin for full induction (A. Gaballa and J. D. Helmann, unpublished data). Very likely, the AraC-type regulator YbtA from the high-pathogenicity island of Yersinia pestis, Yersinia enterocolitica, and Yersinia pseudotuberculosis is also part of this class since it induces the yersiniabactin biosynthesis operon irp21 ybtUTE, the ybtPQXS operon involved in Fe-yersiniabactin uptake and salicylate synthesis, and the Fe-yersiniabactin receptor fyuA (psn) gene and represses its own expression (92). It was found that YbtA binds, possibly as a dimer, to its promoter target sequences independent of yersiniabactin (10). However, as transcriptional activation is yersiniabactin dependent (249), the interaction of the YbtA-DNA complex with Fe-yersiniabactin was proposed to be a prerequisite for the recruitment of the RNA polymerase complex (9).

IrgB, in contrast, is a LysR family-type regulator acting as a transcriptional activator of the Vibrio cholerae IrgA Fe-enterobactin receptor (118). IrgB was not reported to interact with enterobactin or another iron chelator. An unusual type of regulator is the virulence plasmid-encoded Vibrio anguillarum AngR, a Fur-repressed nonribosomal peptide synthetase (NRPS) module-like protein of 120 kDa that acts as an activator of anguibactin biosynthesis and the fatDCBA Fe- anguibactin transport genes at the biosynthetic level and/or as a transcriptional regulator (334). In addition to NRPS condensation, adenylation, and thiolation domains, AngR possesses two helix-turn-helix domains that are preceded by leucine zipper motifs. A point mutation causing a His-to-Asn exchange at position 267, which is located between the first leucine zipper/helix-turn-helix motif, was found to be the cause of anguibactin hyperproduction in the natural V. anguillarum isolate 531A (317). Anguibactin is also involved in the regulation of its uptake since it was found to be an additional inducer of fatDCBA expression (56).

Of these specialized siderophore-dependent regulation systems, the components of ECF sigma factor-dependent regulation, the AraC-type regulators, as well as IrgB and AngR are all regulated by Fur.

Posttranscriptional regulation of bacterial iron homeostasis includes two general mechanisms. In various bacteria, small RNA-targeted mRNA degradation of genes encoding iron-utilizing proteins was observed. The small RNAs involved in this targeting are Fur-regulated antisense RNAs including RhyB in enteric bacteria (205), PrrF1/PrrF2 in P. aeruginosa (335), and, putatively, virulence plasmid pJM1-derived RNAα in V. anguillarum (65). The other mechanism corresponds to posttranscriptional regulation of iron homeostasis in higher eukaryotes. The B. subtilis aconitase CitB is a bifunctional protein with a function analogous to that of mammalian IRP1, a cytosolic aconitase that interacts with regulatory secondary mRNA structures termed iron-responsive elements (IREs) upon losing its [4Fe-4S] cluster during iron deprivation or oxidative stress (283). CitB interacts in an iron-dependent manner with rabbit ferritin IRE as well as IRE-like structures that were found in B. subtilis operons coding for major cytochrome oxidase and Fe-bacillibactin uptake (7).

In yeast, iron homeostasis regulation is also mediated at the transcriptional and posttranscriptional levels. The Saccharomyces cerevisiae transcriptional activator Aft1p and its paralogue Aft2p were shown to bind “iron-responsive” promoter elements during iron starvation (285). Targets of Aft1p/Aft2p regulation include components of the reductive iron assimilatory system such as FET3, FTR1, and the FRE1 to FRE6 genes; the four known Fe-siderophore importers ARN1, TAF1 (ARN2), SIT1 (ARN3), and ENB1 (ARN4); and putative accessory components FIT1 to FIT3 and the low-affinity transporter FET4 (108, 204, 331, 355). Aft1p is constitutively produced, and its function is regulated by its subcellular localization: the protein localizes to the nucleus only if cells are iron depleted (348). Aft1p and Aft2p possess a Cys-X-Cys motif that was suggested to participate in iron-sulfur cluster binding (347) and a nuclear export sequence-like motif. Mutations in either motif cause nuclear retention and constitutive activation of Aft1p (348). Iron-dependent deactivation of Aft1p/Aft2p is abrogated in cells defective for mitochondrial Fe-S cluster biogenesis (55), and iron sensing depends on mitochondrial Fe-S export (286). However, there is no indication that regulation occurs by the direct binding of an Fe-S cluster to the transcription factors, and posttranslational modification might be alternatively involved in Aft1p localization (124). The nuclear monothiol glutaredoxins Grx3 and Grx4 were recently reported to be additional components required for Aft1p iron regulation (238). Further iron-regulatory mechanisms in yeast include the general repressor system Ssn6p-Tup1p, which was shown to regulate the expression of several uptake systems for Fe-siderophores in S. cerevisiae (186), and a global metabolic reprogramming during iron deficiency is achieved by targeted mRNA degradation, which is mediated by Aft1p/Aft2-regulated Cth2p, which is also conserved in plants and mammals (260).

The regulation of siderophore biosynthesis in filamentous fungi was shown to depend on orthologues of the GATA family transcription factor Urbs1, a protein similar to the erythroid transcription factor GATA-1, which was first described for the basidiomycete Ustilago maydis (328). Urbs1 orthologues were also found in ascomycetes such as Sre in Neurospora crassa (358) and SreA in Aspergillus nidulans (126). They are distinguished from all other known fungal GATA factors by the presence of two zinc fingers and a conserved intervening cysteine-rich region, which might represent the most probable binding site for the direct sensing of iron by binding iron or an iron-sulfur cluster (124). While Urbs1 appears to be the exclusive siderophore biosynthesis regulator in U. maydis that represses the siderophore biosynthesis genes sid1 and sid2 during iron repletion (351), additional regulatory factors seem to be present in N. crassa and A. nidulans (124). In A. nidulans, SreA deficiency leads not only to the derepression of siderophore biosynthesis but also to the deregulation of siderophore-mediated iron uptake (236).

In contrast to microorganisms, the regulation of iron homeostasis in mammalians appears to be done exclusively at the posttranscriptional level via the IRP1/IRP2-IRE system (283).

General Steps of Siderophore Pathways

Although the elucidation of new siderophore pathway components has seen much progress during recent years, there is still a substantial discrepancy between information given by the considerable number of well-characterized siderophore biosynthesis systems and the often initially characterized further pathway components. This might be reasonably explained by the fact that siderophore biosynthesis is attractive for semisynthetic drug design combined with various therapeutical applications. However, also, siderophore transport and iron release mechanisms are potentially interesting targets with respect to pathogen control, as discussed below. In this section, siderophore biosynthesis will be treated as a comparative overview of the best-studied systems and their general enzymology. Aspects of siderophore secretion, uptake, and iron release shall then be introduced extensively to the current state of knowledge and with special respect to the latest findings in these growing fields.

Siderophore biosynthesis catalyzed by nonribosomal peptide synthetases.

Depending on the chemical nature of the siderophores, their biosynthesis occurs via different mechanisms. In general, the biosynthesis pathways can be distinguished as being either dependent on or independent of an NRPS. NRPSs represent large multienzyme complexes that activate and assemble a broad array of amino, carboxy, and hydroxy acids, leading to a high structural variability of the generally macrocyclic peptidic products (123). This diversity may be enhanced through various substrate modifications occurring during assembly by the action of specialized domains that are integrated into the standard NRPS domain architecture comprising modular sequences of adenylation (A), thiolation (T, or peptidyl carrier protein [PCP]), and condensation (C) domains. In most cases, the peptide chains are released from the synthetase by an intra- or intermolecular cyclization event catalyzed by commonly C-terminally-located thioesterase (TE) domains (171). NRPSs are responsible mainly for the synthesis of aryl-capped siderophores. NRPS-dependent siderophore biosynthesis in several human pathogens has been elucidated in detail, e.g., enterobactin synthesis in enteric bacteria such as E. coli, Salmonella enterica, Klebsiella spp., and Shigella spp.; yersiniabactin synthesis in Yersinia spp.; pyochelin and pyoverdin synthesis in P. aeruginosa; vibriobactin synthesis in V. cholerae; and mycobactin synthesis in M. tuberculosis (66). Prior to NRPS-catalyzed assembly, the aryl acids 2,3-dihydroxybenzoate (DHB) and salicylate, which are generally used as aryl caps, have to be provided by approaching enzymes. In most bacteria, the genes encoding the NRPS and the enzymes for aryl acid synthesis are directly iron regulated via the Fur repressor. Meanwhile, the enzymes for DHB and salicylate formation as well as several NRPS domains involved in catecholate siderophore assembly have been extensively characterized, and crystal structures are available in many cases. Representative structures of enzymes involved in aryl-capped siderophore biosynthesis are shown in Fig. ​2. The Y. enterocolitica salicylate synthase Irp9 is a homodimer (Fig. ​2A), each protomer of which catalyzes the conversion of chorismate into salicylate via an isochorismate intermediate (162, 163). The X-ray structure of MbtI, the Irp9 homologue in M. tuberculosis and the second known example of a bacterial salicylate synthase, was also recently solved and found to be in a monomeric state (134). Salicylate synthesis in P. aeruginosa was shown to depend on two distinct enzymes, the isochorismate synthase PchA and the isochorismate-pyruvate lyase PchB (104). The activities of both salicylate and isochorismate synthases are highly magnesium dependent, which is also the case for the structurally similar chorismate-utilizing enzymes TrpE, the prototype of anthranilate synthases, and PabB, the aminodeoxychorismate synthase involved in p-aminobenzoate synthesis. In the crystal structure of Irp9 soaked with chorismate, the Mg(II) cofactor was found to be coordinated by two glutamate residues of the active site and the carboxy group salicylate that was found together with pyruvate in the catalytically active crystal, suggesting that this coordination is crucial during catalysis.

FIG. 2.

Structures of proteins involved in siderophore biosynthesis. (A) The Y. enterocolitica salicylate synthase Irp9 homodimer (PDB accession number 2FN1) with reaction products salicylate and pyruvate (shown in red) and the Mg(II) cofactor (shown in yellow)....

The synthesis of DHB from chorismate needs three enzymatic activities. While after isochorismate formation (either as a transient or stable intermediate), during salicylate synthesis, the lyase reaction leads directly to the formation of salicylate when pyruvate is cleaved off, one water molecule is incorporated instead of the pyruvate at the C-3 position during DHB synthesis by the EntB-type isochorismate lyase (or isochorismatase), leading to the formation of a second intermediate, which is 2,3-dihydro-DHB (284). The crystal structure of the E. coli isochorismatase EntB, acting downstream of the PchA-homologous isochorismate synthase EntC (192), was solved recently (81) (Fig. ​2B). In fact, it is a bifunctional enzyme that comprises an N-terminal domain harboring the isochorismatase activity and a smaller C-terminal domain that functions as a PCP-homologous ArCP (aryl carrier protein) domain during enterobactin assembly as discussed below. Both domains are connected via a long proline-rich loop that keeps their active sites at a distance of about 45 Å, thus ensuring the independent activity of both domains. EntB forms a homodimer, and the dimerization takes place via the isochorismatase domain. The last step in DHB synthesis is catalyzed by a 2,3-dihydro-DHB dehydrogenase, a member of the short-chain oxidoreductase enzyme family, which oxidizes the EntB product into the aromatic catechol DHB using NAD+ as a cofactor (289). The crystal structure of the E. coli 2,3-dihydro-DHB dehydrogenase EntA revealed a homotetramer formation (Fig. ​2C), which, as shown previously for other tetrameric members of this enzyme family, is the result of a tight dimer-to-dimer interaction (312). Once synthesized, the aryl acid is used together with further precursors, usually amino acids or polyamines, for siderophore scaffold assembly by the corresponding NRPS. The usually lone-standing aryl acid A domains catalyze the initial step of aryl-capped siderophore assembly by converting either DHB or salicylate in their acyl adenylates. The crystal structure of the B. subtilis DHB adenylation domain DhbE (Fig. ​2D) is an archetype of aryl acid-activating domains (211). Homologous enzymes are the DHB-activating domains EntE and VibE of E. coli and of V. cholerae, respectively, and the salicylate-activating domains YbtE of Yersinia spp., PchD of P. aeruginosa, and MbtA of Mycobacterium species. As a characteristic of NRPS adenylation domains, DhbE possesses a large N-terminal domain that bears the bisubstrate binding site (which is occupied in the depicted structure by DHB-AMP, the native product of catalysis) and a small compact C-terminal “lid” domain that is presented here in superior orientation to the active site, which is the proposed “adenylate-forming conformation.” The N- and C-terminal domains are connected via a short flexible hinge that allows the distal rotation of the lid for substrate binding and product release (“thioester-forming conformation”). Thioester formation occurs upon the adenylation of the aryl acid, which is subsequently tethered to the 4′-phosphopantethein cofactor of an ArCP and is then transferred to the donor position of the first C or Cy (condensation/cyclization) domain. The C-terminal domain of EntB that carries out this function in the enterobactin system bears the covalent attachment site for the cofactor at serine 245, the modification of which converts the domain into its active holo form. Since EntB has to interact with three proteins, which are the phosphopantetheinyl transferase Sfp, the A domain EntE, and the downstream-acting multidomain NRPS EntF, it is a model protein for studying the mechanisms of protein-protein recognition. Mutational studies showed that the interaction with EntE is quite tolerant of a number of point mutations on both the EntB and EntE surfaces (81). The EntB surface for the interaction with EntF, however, was found to comprise three highly conserved hydrophobic residues (M249, F264, and A268) located near the phosphopantetheinylated S245 and in particular on the small helix 3 (Fig. ​2B), suggesting that this structural element is important for the EntB-EntF interaction (179). Homologues of the bifunctional EntB protein exist in B. subtilis (DhbB) and V. cholerae (VibB), bacteria that produce triple-DHB-capped siderophores as well. In contrast, in Pseudomonas spp., Yersinia spp., and Mycobacterium spp., which all produce single-salicylate-capped siderophores, the aryl carrier protein is the first domain in a multidomain NRPS complex (262). The C and Cy domains that catalyze the peptide bond formation between the activated substrates and, in the case of Cy domains, also their cyclization are usually integrated into the NRPS multidomain structure. An exception to this rule is the unusual C-domain VibH catalyzing the first condensation step in vibriobactin synthesis (159), which gave the first crystal structure of an NRPS C domain (160) (Fig. ​2E). VibH couples the thioester-activated DHB with norspermidine, the remaining two amines of which are acylated during following synthesis steps with DHB-methoxazoline-carbonyl moieties that result form cyclizations-condensations of DHB and threonine catalyzed by the multidomain synthetase VibF (158). While VibH is a monomeric pseudodimer, enzymes that are structurally related to NRPS C domains such as chloramphenicol acetyltransferase and dihydrolipoamide acetyltransferase are known to be homotrimers.

As vibriobactin synthesis demonstrates, the Cy domain-catalyzed formation of intramolecular heterocycles takes place in various aryl-capped siderophores. Anguibactin, pyochelin, and yersiniabactin contain thiazoline rings resulting from the cyclization of cysteine side chains (106, 264). In-trans-acting reductase domains catalyze the subsequent conversion of thiazolines into thiazolidines during pyochelin and yersiniabactin synthesis (217, 243). In mycobactins and structurally related mixed aryl-capped hydroxamate siderophores such as acinetobactin and frankobactin, oxazoline rings are found as a result of serine or threonine cyclization (341), and in the case of heterobactin A, a hydroxybenzoxazole ring is built up during the assembly process (45). In some biosynthesis systems, polyketide synthase (PKS) domains are part of the siderophore assembly lines. High-molecular-weight protein 1, involved in yersiniabactin synthesis, is an NRPS/PKS hybrid synthetase, the PKS domains of which introduce a malonyl moiety into the nascent peptide chain and perform subsequent bismethylation and β-ketoreduction on this building block (217). During mycobactin synthesis, a β-hydroxyacyl is incorporated by the NRPS/PKS hybrid MbtB and the MbtC and MbtD PKSs (263). Deletion of MbtB, which additionally catalyzes phenyloxazoline formation between salicylate and serine, completely disrupts salicylate-derived siderophore synthesis in M. tuberculosis, leading to reduced growth in iron-limited medium and in macrophage-like THP-1 cells (80). Mycobactins are produced by enzymes of the mbt-1 and mbt-2 gene clusters, the latter of which was recently found to comprise the four tailoring enzymes for mycobactin acylation (178). Mycobactins contain a long lipid chain that renders them lipid soluble and favorable for associations with the mycobacterial cell envelope. Pathogenic mycobacteria such as M. tuberculosis and Mycobacterium leprae, however, apparently use the same genes to synthesize mycobactin derivatives called carboxymycobactins, which possess a shorter carboxyalkyl chain that renders them more hydrophilic with respect to their function as extracellular siderophores (268). However, it was recently shown that lipophilic mycobactins can also serve as extracellular siderophores within mycobacterium-colonized macrophages by diffusing through the membrane of the occupied macrophage phagosomes and, after scavenging iron from the intracellular pool, accumulating in intracellular lipid droplets that are delivered to the phagosomes by lipid trafficking (201). Nonpathogenic mycobacteria such as Mycobacterium smegmatis and Mycobacterium neoaurum use, in addition to carboxymycobactins, non-aryl-capped exochelins as extracellular siderophores, which are predicted to be synthesized by the FxbB and FxbC NRPSs (350, 360). Product release from the NRPS multienzyme complex is, in the majority of cases, catalyzed by a C-terminus-standing TE domain, and this final reaction step often leads to the concomitant formation of macrocyclic structures if side chains of the assembled peptide are used for the nucleophilic attack at the TE-bound ester intermediate. NRPS-dependent siderophore macrocyclization has so far been reported for enterobactin and bacillibactin that contain trilactone backbones resulting from iterative cyclization catalyzed by the C-terminal TE domains of the EntF and DhbF assembly lines, respectively (212, 300). During this cyclotrimerization reaction, three peptide chain intermediates, either DHB-seryl or DHB-glycine-threonyl, are successively assembled via ester bond formation between the hydroxy groups of the seryl or threonyl residues of the chain tethered to the TE and the electrophilic thioester of the following chain, which is tethered to the PCP upstream of the TE. NRPS-dependent cyclization reactions are also putatively involved in the synthesis of the cyclopeptide backbones of pyoverdins, alterobactin A, and fungal ferrichrome-type siderophores.

Finally, the optimization of NRPS-dependent peptide siderophore synthesis is mediated in several systems by trans-acting type II thioesterases, which have a general editing function by catalyzing the deacylation of misprimed or misacylated PKS acyl carrier proteins and NRPS ArCPs or PCPs (294). Siderophore biosynthesis proofreading was reported for the type II thioesterases PchC and EntH during pyochelin and enterobactin formation in P. aeruginosa and E. coli, respectively (D. Leduc and E. Bouveret, unpublished data; 274). While PchC proved specific for regenerating the PCP domains of PchE and PchF that were not correctly charged with l-cysteine but were charged with its analogue 2-aminobutyrate, EntH was shown to interact specifically with the ArCP of EntB and to regenerate the domain when mischarged with salicylate. Thioesterase-dependent NRPS (and PKS) editing might be suspected in further siderophore synthesis systems that bear lone-standing thioesterases such as yersiniabactin synthesis cluster-encoded YbtT, which was found not to be necessary for both product release and full in vitro activity of the yersiniabactin assembly machinery (217), or such as Streptomyces coelicolor CchJ, which is required for coelichelin biosynthesis in vivo and hence was provisionally suggested to be a thioesterase for hydrolytic product release rather than for assembly line editing (182).

In some cases, NRPSs are partially involved in the synthesis of hydroxamate and carboxylate siderophores to build a peptidic backbone to which the iron-coordinating residues are attached. This has been reported for coelichelin of S. coelicolor (52, 182), the pyoverdins of fluorescent Pseudomonas spp. (2, 224), the exochelins from nonpathogenic mycobacteria (350, 360), and the ferrichromes/ferricrocins from various fungi (84, 295, 333, 351) and can be anticipated for alterobactin of Alteromonas luteoviolacea, the pseudobactins of Pseudomonas spp., the azotobactins of Azotobacter spp., and the ornibactins of Burkholderia spp.

Siderophore biosynthesis independent of nonribosomal peptide synthetases.

Hydroxamate and carboxylate siderophores are assembled by NRPS-independent mechanisms in the majority of cases. The synthesis of siderophores belonging to these two main classes commonly relies on a diverse spectrum of enzymatic activities such as monooxygenases, decarboxylases, aminotransferases, ac(et)yltransferases, amino acid ligases, and aldolases (51). Siderophores synthesized by NRPS-independent pathways are found as virulence factors in several pathogens, e.g., aerobactin in enteric bacteria (114), alcaligin in B. pertussis and B. bronchiseptica (220, 234), staphylobactin in Staphylococcus aureus (68), and petrobactin (formerly anthrachelin) in Bacillus anthracis (48, 174). Among the NRPS-independently assembled siderophores, petrobactin represents an exception because it is capped with two catecholate moieties for iron coordination (see Fig. ​1 for structure). However, the use of 3,4-dihydroxybenzoate moieties is so far unique in the structural world of siderophores, and a possible explanation for their utilization is given in the section dealing with pathogen-host interactions below.

In general, hydroxamate moieties are built in two steps. The first reaction step is an N-hydroxylation catalyzed by reduced flavin adenine dinucleotide (FAD)-dependent monooxygenases that use molecular oxygen and a set of amino acids and polyamines as substrates. In most of the known pathways, one oxygen atom is transferred either to the ɛ-amino group of lysine (aerobactin pathway), to the δ-amino group of ornithine (ferrichrome/ferricrocin, coprogen, rhodotorulic acid, and fusarinine pathways), or to one amino group of the corresponding decarboxylation products cadaverine (desferrioxamine E and, putatively, bisucaberin pathways) and putrescine (alcaligin and, putatively, putrebactin pathways), respectively (51, 341). In the rhizobactin 1021 pathway, the unusual diamine 1,3-diaminopropane is suggested to be the substrate for N-hydroxylation (203). In contrast to the NRPS-independently catalyzed sequence of hydroxamate siderophore assembly, the introduction of hydroxamate functions into the NRPS-derived mycobactin scaffold was concluded to be the final synthesis step due to the detection of didehydroxymycobactins in early-infection cultures of M. tuberculosis (219). In this case, the monooxygenase MbtG catalyzes N6-hydroxylation of two lysines that are already integrated into the complex siderophore scaffold (178).

The formylation (leading to free hydroxamic acid moieties) or acylation of the hydroxylated amine generally represents the second step yielding the functional hydroxamate and is catalyzed in the case of acylation by acyl coenzyme A transferases. Formylated N5-hydroxy-ornithines are present in pyoverdins and in ornibactin. Acylation of hydroxylated amines is much more frequent. As substrates, coenzyme A derivatives of various carboxy acids such as acetate (aerobactin), succinate (desferrioxamine E), β-hydroxybutyrate (pyoverdins and ornibactin C4), or decenoate (rhizobactin 1021) are used. In mycobactin synthesis, however, the acylation is proposed to occur prior to the N-hydroxylation since the cyclization of the C-terminal lysine, which might be catalyzed by some kind of thioesterase or condensation domain (66), was already found in isolated didehydroxymycobactins (219). The free (α-hydroxy-)carboxylate moieties in (α-hydroxy-)carboxylate siderophores may be derived from hydroxy-carboxylic acids such as citrate (staphyloferrin A and vibrioferrin), 2-oxo-glutarate (achromobactin and vibrioferrin), and malonic acid (rhizobactin DM4) or amino-carboxy acids such as alanine in rhizobactin DM4 or 2,3-diaminopropionic acid in staphyloferrin B, which are often (but not always) coupled to the backbone by ester or amide bonds. Generally, the ligation of citrate occurs via one of its prochiral carboxyl groups, while 2-oxo-glutarate is attached via its C-5 carboxyl group (51). Free α-hydroxy-carboxylate moieties may also result from introducing β-hydroxy-aspartates in a peptidic backbone sequence such as in pyoverdins and alterobactin.

The final ligation of the building blocks in NRPS-independent siderophore assembly is catalyzed by NTP-dependent siderophore synthetases of the IucA type and/or the IucC type mainly by amide bond formation and sometimes also via ester linkages such as those in achromobactin or vibrioferrin. IucA and IucC are the prototypes of NRPS-independent siderophore synthetases that catalyze the penultimate and the final steps in aerobactin synthesis, respectively, and all known NRPS-independent siderophore synthesis pathways utilize at least one enzyme with high sequence similarity to the aerobactin synthetases (51).

Siderophore secretion.

Siderophore secretion systems have been identified in only a few microorganisms so far. The exporters that were found or suggested to be involved in siderophore release belong to efflux pumps of the major facilitator superfamily (MFS); the resistance, nodulation, and cell division (RND) superfamily; and the ATP-binding cassette (ABC) superfamily.

The MFS is a large and diverse superfamily of transporters that carry out uniport, solute:cation (proton or sodium) symport and/or solute:proton or solute:solute antiport (287). They exhibit specificity for a broad range of compounds including various primary metabolites, drugs, neurotransmitters, siderophores (efflux), iron-charged siderophores (see “Uptake of siderophore-delivered iron”), and organic and inorganic anions. They possess either 12, 14, or 24 α-helical transmembrane segments (TMSs). In contrast, members of the RND superfamily probably all catalyze substrate efflux via a proton antiport mechanism (320). Generally, they possess a single transmembrane-spanning region at the N terminus that is followed by a large extracytoplasmic region and then six additional transmembrane regions, a second large extracytoplasmic section, and five final transmembrane regions at the C terminus. Substrates of RND efflux pumps are heavy metals, various drugs (e.g., tetracycline, chloramphenicol, fluoroquinolones, and β-lactams), lipooligosaccharides, lipids, pigment(s), siderophore(s), and possibly sterols in eukaryotes. RND members of several families act in conjunction with a membrane fusion protein (MFP) and an OM factor (OMF) to yield efflux across both membranes of the gram-negative bacterial cell envelope in a single energy-coupled step. Both MFS and RND superfamily members are found ubiquitously in all three kingdoms of life.

The MFS involved in enterobactin secretion in E. coli is a 12-TMS protein termed EntS that is encoded in the Fur-regulated ent-fep gene cluster (102). An entS mutant shows significantly reduced secretion of enterobactin but increased release of enterobactin breakdown products, which was explained by Fes-catalyzed intracellular enterobactin hydrolysis and the subsequent diffusion and/or release of the breakdown products by further cytoplasmic export systems. Thus, while export across the cytoplasmic membrane (CM) is mediated primarily (but not exclusively) by EntS, the OM channel protein TolC was shown to be exclusively responsible for enterobactin release across the OM (23). Altogether, there are a couple of possibilities for how enterobactin export is committed, which can be addressed either as a one-step or as a two-step scenario. As a one-step process, enterobactin export may involve a tripartite transport system composed of a CM transporter (primarily EntS but putatively at least one more efflux pump), an MFP (yet unknown), and TolC as an OMF. In a possible two-step process, enterobactin could be released by EntS (and putatively another CM transporter) into the periplasm, from where clearance is mediated by a TolC-comprising RND-type transporter. Such a two-step process was recently shown for heavy metal efflux (228). As a third alternative, a TolC-dependent but EntS-independent export system(s) that transports enterobactin from both the cytoplasm and the periplasm to the environment might exist. Enterobactin breakdown products, however, were also released through the OM in a tolC mutant (23), suggesting that there are further OM gates for clearing the periplasm from catecholate fragments.

MFS efflux pumps that possess 12 membrane segments are involved in siderophore export in other bacteria. In most cases, they are Fur-dependently regulated. Legiobactin, a siderophore of yet unknown structure produced by Legionella pneumophila, is exported by the MFS exporter LbtB (8). LbtB is encoded by the second gene of the lbt operon that also encodes the IucA-IucC-related siderophore synthetase LbtA and the LbtB-like transmembrane protein LbtC, which, however, is not involved in legiobactin efflux. The Erwinia chrysanthemi and the Vibrio parahaemolyticus hydroxycarboxylate siderophores achromobactin and vibrioferrin are exported via the YhcA and the PvsC MFS exporters encoded within the corresponding siderophore biosynthesis operons, respectively (314). The CsbX MFS exporter encoded upstream of the catecholate siderophore biosynthesis (cdb) operon of Azotobacter vinelandii facilitates the secretion of catecholate siderophores related to protochelin (240). The AlcS MFS exporter of B. pertussis and B. bronchiseptica that mediates the export of the cyclic hydroxamate siderophore alcaligin is, in contrast, constitutively produced and, as it regulates the intracellular alcaligin concentration, is part of the alcaligin/AlcR-dependent regulatory circuit controlling alcaligin gene expression (35). An alignment of the six MFS efflux proteins involved in siderophore secretion identified so far (Fig. ​3) reveals low protein sequence identities (below 20%). However, this might be expected since neither the phylogenetic relationship of the bacterial sources (except for E. coli and E. chrysanthemi) nor the structural relationship of the transported siderophores (if the structure is known at all) is very high. Generally, members of the MFS do not possess such well-defined conserved motifs as it is known from other transporter superfamilies, e.g., those of the ABC type. The conserved 13-amino-acid (aa) motif (GX2ADRYGR[R/K] [R/K]X[L/I]; boldface indicates conservation higher than 50% in the alignment shown in Fig. ​3) found in the alignment between TMS2 and TMS3 is very similar to the major consensus motif described for the MFS in this sequence region (241). This motif is thought to be involved in promoting conformational changes in the protein upon substrate binding, allowing the trafficking of substrates through the membrane, and may also act as a gate for substrate transport regulation (308). Further studies on the growing number of MFS-type siderophore exporters should provide deeper insights into transport type, affinity, and specificity.

FIG. 3.

Alignment (ClustalW, shown in BOXSHADE format) of MFS-type proteins involved in the secretion of siderophores. Sections of predicted TMSs are shown individually for each protein and are indicated with red letters and/or red shading. Nonshaded letters...

The P. aeruginosa efflux system MexA-MexB-OprM represents a typical RND exporter of gram-negative bacteria consisting of an MFP (MexA), an RND efflux pump with 12 TMSs (MexB), and an OMF (OprM) (189). Since the putative operon of this efflux machinery is iron regulated and since a mutant strain was growth sensitive in dipyridyl-containing iron limitation medium, it was suggested that this exporter is involved in the efflux of pyoverdin, the primarily virulence-associated siderophore of P. aeruginosa (257). Several drugs that are exported by MexA-MexB-OprM have structures related to the pyoverdin chromophore and are capable of binding iron. However, data showing a reduction of pyoverdin secretion in the exporter mutant strain have not been reported so far.

The ABC-type transporter superfamily contains both efflux and uptake transport systems that generally mediate transport via ATP hydrolysis without protein phosphorylation (28, 69, 72, 152). They consist of two integral membrane domains and two cytoplasmic domains (ABC subunits) for NTP (usually ATP) binding and hydrolysis. Both the transmembrane and the cytoplasmic domains may be present as homodimers or heterodimers. While uptake porters generally have their domains as distinct polypeptide chains, efflux systems usually have them fused. ABC-type efflux systems are abundant in both prokaryotes and eukaryotes. The eukaryotic efflux systems often have the four domains (two cytoplasmic domains representing the nucleotide-binding folds [NBFs] and two integral membrane domains) fused into either one or two polypeptide chains, while in prokaryotes, they are generally fused into two chains, both of them containing one integral membrane channel constituent and one cytoplasmic ATP-hydrolyzing constituent. Generally, the integral membrane domains of the ABC-type efflux systems possess six α-helical TMSs each. Representative of the structural topology of bacterial ABC-type exporters are the crystal structures of the open and the closed conformations of the E. coli and V. cholerae MsbA multidrug transporter homologues, respectively (53, 54), and the structure of the S. aureus Sav1866 multidrug transporter (70). Based on different structural aspects, two mechanistic models of substrate translocation, involving either a sideward-directed movement of the subunit halves (275) or domain swapping, which is mediated by twisting the protomer subunits (70), have been proposed. Cross-linking studies with the human multidrug resistance P glycoprotein suggested another model proposing a substrate-induced conformational change in the TMSs of the transporter (145, 197).

A mutagenesis study of M. smegmatis led to the identification of the IdeR-regulated exiT locus encoding an ABC-type exporter that is responsible for the secretion of exochelin MS (360), which belongs to the group of water-soluble extracellular hydroxamate siderophores produced by nonpathogenic mycobacteria. ExiT, which is encoded upstream of the exochelin biosynthesis genes fxbB and fxbC, possesses a C-terminally-located NBF and eight proposed TMSs that were suggested to be expanded up to 12 due to the length of three of these segments. Although fusions of two transporter half-segments leading to ABC-type exporters comprised in a single tetradomain polypeptide chain are observed in bacteria, the second NBF is missing in the ExiT sequence. A closer examination reveals that six of the eight proposed TMSs cluster adjacent to the C-terminal ABC, and the two remaining segments are found at the N terminus, leaving a large hydrophilic region of 430 aa between the TMS regions. It was speculated that these features might be an adaptation to the unique architecture of the mycobacterial cell envelope (360). In eukaryotes, the group of multidrug-resistance-related protein-like ABC-type transporters is known to possess an additional trans-membrane-spanning domain of approximately 200 aa at the N terminus of the protein; however, these proteins have a TMSn[TMS6-NBF]2 topology and a characteristic “regulatory” or “connector” domain between their homologous halves (308), both of which are not characteristic for ExiT. Since numerous possible start codons were found in the exiT open reading frame and since the reported protein sequence was based on the farthest upstream-located translation initiation site that was tentatively chosen, the putative N terminus of ExiT might be significantly shorter, thus leading to an ExiT protein with a more common bacterial ABC-type exporter architecture.

Another ABC-type transport system has recently been suggested to function in the secretion of salmochelins (191), which are glucosylated derivatives of enterobactin produced by pathogenic E. coli and Salmonella strains. The iroA gene cluster harbors the gene iroC that encodes a four-domain ABC-type fusion protein with [TMS6-NBF]2 topology showing about 30% identity to eukaryotic P glycoproteins involved in multidrug resistance (15, 16, 132). Genome database searches reveal that there are only few bacterial species harboring four-domain ABC-type fusion proteins, and as in the case of IroC-harboring strains, they often live in close contact to eukaryotes either as pathogens (opportunistic pathogens such as Nocardia farcinica or obligate pathogens such as M. tuberculosis or C. diphtheriae) or as symbionts (such as Frankia alni). Thus, it might be speculated that the prokaryotic development of such fusion types has been advanced by interspecies gene transfer. Although the similarity of IroC to ABC-type efflux proteins is significant, growth promotion studies indicated that IroC might be responsible for the uptake of linearized forms of Fe-salmochelin and Fe-enterobactin (359). Thus, two conflicting models of salmochelin utilization currently exist, one assigning IroC as a salmochelin exporter (95, 191) and the other one assigning IroC as an importer of the iron-charged linearized salmochelin trimer (359). IroC-dependent Fe-salmochelin transport studies or iroC mutant phenotype analyses might help to clarify the opposing interpretations.

Interestingly, during investigations of phenotypes of several exporter mutants, it was observed that they either accumulate (35) or do not accumulate (102, 360

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