Siderophore cycling has two components: (1) the export of siderophores and (2) the import of the iron-loaded siderophore [
88]. Presumably, siderophores are recycled and used multiple times by the producing organism or community. The extent of recycling is an intractable problem experimentally, and it is thus often only stated that siderophores are recyclable, without any investigation of the actual cycling. In the case of photoreactive siderophores such as synechobactin, the chemical change induced by light permanently changes the siderophore, making its recycling difficult to envisage. In the case of more complex siderophores such as anachelin, the energetic investment per molecule makes recycling more likely. A scheme showing the overall cycling of siderophores in a typical organism is shown in
Figure 4.
5.2. Siderophore Import
The import of iron-loaded siderophores is broadly similar across many bacteria and is illustrated in
Figure 4. The transport of incoming siderophore-iron complexes into the periplasmic space is enabled by receptor proteins, TonB-dependent transporters (TBDT) located in the outer membrane. TBDTs consist of a β-barrel domain and a “plug” domain in the barrel interior that acts in concert with a TonB-box on the periplasmic side. Energy for transport is derived from the proton-motive force and is mediated through the association of TonB with inner membrane proteins ExbB and ExbD, forming a TonB-ExbB-ExbD-complex. Once in the periplasm, Fe
3+-loaded siderophores are transported across the inner membrane, often by ABC-transporters [
90].
Once inside the cell, iron has to be removed from the siderophore. This can happen either through the reduction of iron from Fe
3+ to Fe
2+ by ferric siderophore reductases or by ferric-siderophore hydrolases [
89] or by the photolysis of iron-bound siderophores, as described for synechobactin. In the case of photolysis, whether the reduction event takes place outside or inside the cell is not clear.
In
Anabaena sp. PCC 7120, the import of iron complexed schizokinen across the outer membrane is accomplished by two TBDTs, named the schizokinen transporter (
alr3097, SchT) [
49] and IutA2 (
alr2581). Transcription data indicate that IutA2 is transcribed earlier in the iron starvation response than SchT [
50].
The TBDTs are likely dependent on the inner membrane complex TonB3-ExbB3-ExbD3 (
all2585-all5047-all4056) for energy. The genes are upregulated under an iron limitation, while similar genes show distinct regulation pattern under other conditions. Additionally, deletion mutations of the genes show an iron starvation phenotype [
88].
In the periplasm, Fe-schizokinen is recognized and transported to the cytoplasm via the hydroxamate system FhuBCD (
all0387,
all0388, and
all0389, respectively). These genes are similar to FhuBCD in
E. coli, where FhuD functions as a periplasmic binding protein, FhuB is a membrane-embedded transporter and FhuC is an ATP-binding protein. Interestingly, while all three genes are upregulated under an iron limitation, they are regulated independently, as
fhuC responds differently than
fhuB/D to changing copper and citric acid levels. The significance of this is not clear [
50,
88].
The release mechanism for ferric iron bound to schizokinen has been found to involve a ferric siderophore reductase in other bacteria [
91].
TBDTs are unevenly distributed in cyanobacteria. In general, TBDTs seem to outnumber the amount of TonB-proteins, indicating some functional flexibility in TonB-complexes. The genome of
Anabaena sp PCC 7120 contains 22 TBDTs, while no candidate genes were identified in the marine
Prochlorococcus. Synechococcus sp PCC 7002 contains 6 TBDTs, with two found by sequence similarity to resemble known schizokinen transporters, and two found to resemble hydroxamate transporters more broadly [
92]. As TBDTs function in the uptake of a variety of compounds [
90], variability in TBDTs cannot be explained purely in terms of siderophore or iron uptake more broadly.
The large number of TBDTs in some organisms is partially explained by the possibility of uptake of siderophores which are not produced by the organism (xenosiderophores), known as siderophore piracy.
Anabaena sp PCC 7120 has been shown to make use of siderophores such as aerobactin [
24] and the tris-hydroxamate desferroxamine B (DFB). Aerobactin is likely taken up by the same TBDTs as schizokinen, but DFB is more dissimilar to schizokinen and is transported across the outer membrane by an unidentified TBDT. The import of different siderophores appears to converge on the use of FhuBCD across the inner membrane [
50,
93].
Similarly, the non-siderophore-producing
Synechocystis sp. PCC 6803 is capable of utilizing both schizokinen and the unnamed siderophore of
Anabaena variabilis ATCC 29413 and can grow with iron-siderophore complexes as their only source of iron. While reductive iron uptake is considered the primary means of iron acquisition for this organism, the genome of
Synechocystis has been found to code for a singular
tonB (slr1484) gene, which functions along with exbB1–exbD1 (
sll1404, sll1404) in apparently typical siderophore uptake [
94]. Additionally, siderophore uptake in
Synechocystis sp. PCC 6803 is dependent on TBDT SchT (
sll1206), which is organized in a gene cluster along with the gene coding for the ABC-type transporter FecB1CDE (
slr1316 to
slr1319). This system is able to operate independently of the ferric and ferrous iron transporters that are presumed to be the main iron acquisition mechanisms in
Synechocystis sp. PCC 6803 [
95].