The structure of anguibactin is unique in containing both catechol and hydroxamate metal-chelating functional groups (Actis et al., 1986), derived from 2,3-dihydroxybenzoic acid (DHBA) and N-hydro-histamine, respectively. Utilizing the chorismate, plasmid-carrying V. anguillarum strain 775 generates a repertoire of molecules through a ribosome-independent process and finally synthesizes anguibactin. This biosynthesis is controlled by a number of genetic determinants. To date, more than 10 different genes have been described and yield anguibactin-related phenotypes in plasmid-carrying V. anguillarum strains when mutated by genetic approaches (Table S1). Most are located on the plasmid pJM1 or pJM1-like plasmids while some are on the chromosomes (Figure 1). First, chorismate is catalyzed stepwise by a series of proteins, AngC/VabC (isochrorismate synthase), AngB/VabB (isochrorismatase; Du et al., 2017), and VabA (2,3-dihydro-2,3-dehydroxybenzoate dehydrogenase), to synthesize DHBA (Figure 2; Alice et al., 2005; Balado et al., 2008). In later steps, phosphopantetheine transferase AngD is required to transfer a phosphopantetheinyl moiety to a serine residue of AngB and AngM (Balado et al., 2008). AngE/VabE (2,3-dehydroxybenzoate-AMP ligase) activates DHBA to form acyl adenylate and further transfers it to the free thiol of the phosphopantetheine AngB (Liu et al., 2004; Alice et al., 2005). AngC, AngB, and AngE are pJM1-encoded proteins while VabC, VabB, VabA, and VabE are chromosomally encoded with VabC, VabB, and VabE showing functional redundancy with AngC, AngB, and AngE, respectively. AngN catalyzes this DHBA thioester to combine with cysteine, which is activated by AngR and tethered by AngM, thereby producing a dihydroxyphenyl-thiazoline-thioester. AngH (histidine decarboxylase; Tolmasky et al., 1995; Barancin et al., 1998) and possibly AngU (Naka et al., 2013b) modify the histidine to form N-hydro-histamine, which is then transferred to dihydroxyphenyl-thiazoline-thioester to yield anguibactin. It is interesting to note that homologs of these plasmid-located genes are all present on the chromosome of Vibrio harveyi, which is also able to produce anguibactin, suggesting that the plasmid-mediated anguibactin system might originate from V. harveyi or vice versa (Naka et al., 2013a,b).

Plasmidless O1 strains and those belonging to a number of other V. anguillarum serotypes synthesize a chromosome-mediated siderophore, vanchrobactin (Lemos et al., 1988; Soengas et al., 2008). In a similar way to anguibactin synthesis, DHBA of these V. anguillarum strains is also produced from chorismate by the sequential activities of VabC, VabB, and VabA. However, how vanchrobactin is synthesized from the DHBA precursor remains obscure, even though some genes have been found to be indispensable for this process, including vabB, vabD, vabE, and vabF (Balado et al., 2006, 2008). According to well-studied pathways for synthesis of anguibactin and vibriobactin, a siderophore produced by Vibrio cholerae, late steps for vanchrobactin formation in V. anguillarum have been proposed during which DHBA is assembled (Figure 3; Balado et al., 2006). Specifically, VabD contributes to transfer of a phosphopantetheinyl moiety to the aryl carrier domain of VabB and the peptidyl carrier domain of VabF, respectively. Like anguibactin formation, VabE activates DHBA and arginine to yield acyl adenylates, and then delivers them to VabB and VabF, respectively. The DHBA-VabB is combined with arginine by the condensation domain of VabF to form (2,3-dihydroxybenzoyl)argininate. Finally, the condensation domain of VabF in (2,3-dihydroxybenzoyl)argininate-VabF may be loaded with VabE-activated serine, which adheres to the peptidyl carrier domain of VabF to produce vanchrobactin. Although similar roles of AngB and VabB are proposed in these processes, the aryl carrier protein (ArCP) domain of VabB is not able to complement the function of the AngB ArCP domain (Di Lorenzo et al., 2011), suggesting different but unknown roles may occur between AngB and VabB during anguibactin and vanchrobactin formation.

Once siderophores are produced and exported to the environment, they capture iron to form a ferric-siderophore complex, which is recognized by a specific transporter on the surface of the outer membrane. In V. anguillarum, ferric-anguibactin is translocated across the outer membrane via its specific transporter FatA, which is essential for ferric-anguibactin uptake (Walter et al., 1983; Actis et al., 1988; Lopez and Crosa, 2007; Lopez et al., 2007). This process requires the TonB2 system for energy transmission, which originates from the proton-motive force of the inner membrane (Stork et al., 2004).

The TonB2 complex is located across the inner membrane and comprises TonB2, ExbB2, ExbD2, and TtpC (TonB2 complex-associated transport protein C), all of which are indispensable for ferric-anguibactin import to the periplasmic space. Deletion of either of these genes completely abolishes ferric-anguibactin uptake, and they are thereby considered essential virulence factors for V. anguillarum since ferrisiderophore transport during iron uptake is a critical step for virulence (Stork et al., 2004, 2007). Notably, TtpC which shows homology to the TolR protein of E. coli, has been identified to be part of the TonB2 system in several Vibrio species, including V. anguillarum (Stork et al., 2007), V. cholerae (Stork et al., 2007), Vibrio alginolyticus (Wang et al., 2008), Vibrio parahaemolyticus (Kuehl and Crosa, 2010), and Vibrio vulnificus (Kuehl and Crosa, 2009). Despite a high similarity among all pathogenic vibrios studied thus far, the TtpC proteins likely play an important role in specific iron transport mediated by the TonB2 system, a conclusion drawn based on the observation that V. anguillarum ΔttpC cannot be complemented by the TtpC protein from V. cholerae (Stork et al., 2007; Kuehl and Crosa, 2009).

In addition to anguibactin transport, the TonB2 system is also involved in the uptake of vanchrobactin and the xenosiderophore (siderophore produced by other organisms) enterobactin. The transport of vanchrobactin and enterobactin is mediated by another outer membrane transporter, FvtA, which displays vanchrobactin-dependent expression (Balado et al., 2008, 2009; Naka et al., 2008). In the serotype O2 V. anguillarum strain RV22, loss of fvtA leads to impaired growth under iron-limiting conditions due to incapability of vanchrobactin transport in cells (Balado et al., 2009). Besides FvtA, during transport of the xenosiderophore enterobactin, it seems another transporter, FetA, is present that shows specific binding to enterobactin, and the expression of FetA is regulated by FetR (Naka and Crosa, 2012). This fetA-fetR cluster in V. anguillarum is speculated to derive from V. cholerae based on similar molecular organization and high similarity (Naka and Crosa, 2012).

Subsequently, when combined with the periplasmic lipoprotein FatB, ferric-anguibactin passes through the cytoplasmic membrane by using an ATP-binding cassette (ABC) transporter, which includes inner membrane permeases consisting of a heterodimer of FatC and FatD, and an ATPase FatE (Köster et al., 1991; Actis et al., 1995; Naka et al., 2010, 2013c). FatBCD are required for ferric-anguibactin transport while a fatE mutant is still capable of ferric-anguibactin transport due to the presence of its homolog FvtE (Naka et al., 2013c). Double deletion of fatE and fvtE significantly impairs ferric-anguibactin uptake (Naka et al., 2013c). Similarly, the passage of vanchrobactin through the inner membrane is also achieved in a stepwise manner by a periplasmic protein, FvtB, and an ABC transporter, FvtCDE, which together are essential for ferric-vanchrobactin or ferric-enterobactin import; deletion of either protein causes a defect in ferric-vanchrobactin and ferric-enterobactin transport (Naka et al., 2013c). Different from the specific involvement of FatE in the uptake of anguibactin, FvtE can transport both anguibactin and vanchrobactin/enterobactin (Naka et al., 2013c).

It is noteworthy that although fatDCBA encoding for ferric-anguibactin transport are located on the pJM1-type plasmid in serotype O1 strains, they are also present on the chromosomes of plasmidless vanchrobactin-synthesizing strains, albeit fatA is inactivated by an insertion sequence (Bay et al., 2007; Balado et al., 2009). In fact, gene clusters coding for vanchrobactin synthesis and uptake are ubiquitous in both anguibactin- and vanchrobactin-producing V. anguillarum strains (Balado et al., 2009), while plasmid-harboring strains do not produce vanchrobactin due to an inactivated chromosomal vabF gene (Figure 1; Naka et al., 2008). This is caused by the presence of the insertion sequence RS1, observed in all serotype O1 strains carrying a pJM1-type plasmid, and when RS1 is removed, pJM1-containing strains can simultaneously produce the siderophore vanchrobactin (Naka et al., 2008). However, because anguibactin appears to have a higher affinity for iron than does vanchrobactin, it is suggested to be the primary siderophore for iron import even though vanchrobactin is produced in the cell (Naka et al., 2008). Nevertheless, the minimal inhibitory concentration (MIC) values for iron chelators in plasmid-bearing strains seem to be lower than those of plasmidless V. anguillarum strains, which might be caused by higher stoichiometry of vanchrobactin in the plasmidless strains (Conchas et al., 1991).

Heme usually exists abundantly but not freely as a chemical compound in host cells. Therefore, bacteria secrete exotoxins such as cytolysins, proteases, or hemolysins to release it for further uptake. In V. anguillarum, several hemolysin genes have been identified and some of them are involved in V. anguillarum virulence (Hirono et al., 1996; Rodkhum et al., 2005; Rock and Nelson, 2006; Li et al., 2008, 2013; Xu et al., 2011). After its release, free heme is captured by a putative outer membrane transporter, HuvA, which has been shown to be essential for heme uptake because the huvA mutant does not grow when heme is used as the sole iron source (Mazoy et al., 2003). However, compared with the wild type, the huvA mutant still maintains heme-binding activity under the conditions tested (Mazoy et al., 2003), indicating that HuvA is not the only protein capable of binding heme. In line with this, two heme-binding proteins with molecular masses of 39 and 37 kDa were isolated from V. anguillarum serotype O1 and O2 strains, respectively, which are completely different from the 79 kDa HuvA protein (Mazoy and Lemos, 1996b; Mazoy et al., 1996). Therefore, Mazoy et al. speculate that, besides HuvA, additional proteins are probably present in V. anguillarum and function in heme binding, but not in its transport (Mazoy et al., 2003; Lemos and Osorio, 2007). Like siderophore import, this process is also energy-dependent, where HuvA is energized by TonB systems. The difference is that not only is the TonB2 complex involved in heme utilization, but a TonB1 system composed of TonB1-ExbB1-ExbD1 is also available for energy supply (Stork et al., 2004). Deletion of all genes for these two TonB systems leads to a complete defect in heme uptake and thus avirulence to the host, suggesting a key role of TonB systems in V. anguillarum virulence (Stork et al., 2004). The recent structural studies of the Ton complex from E. coli provide a mechanistic insight into this complex (Celia et al., 2016). It is proposed that the functional unit of the Ton complex contains an ExbB pentamer, an ExbD dimer, and at least one TonB. Electrophysiology experiments suggest that the ExbB-ExbD forms pH-sensitive channels, by which the Ton complex likely harnesses the proton-motive force for energy production and transduction.

Bioinformatic studies indicate that genes of the two TonB systems exist ubiquitously among all Vibrio species, and moreover, a third TonB system is even observed in some vibrios and other marine organisms (Kuehl and Crosa, 2010; Kustusch et al., 2011). However, it remains unclear why so many TonB systems are present in the Vibrio species. The V. anguillarum strains lack HuvA but contain an alternative heme transporter, HuvS, which is able to restore heme transport in a huvA mutant (Mouriño et al., 2005). The observation that huvS and huvA possess similar flanking DNA sequences implies that horizontal transmission and recombination might have occurred and thus be responsible for this genetic diversity (Mouriño et al., 2005).

Furthermore, the huvA gene is located in a gene cluster coding for nine heme uptake-related proteins, including HuvA, HuvZ, HuvX, TonB1, ExbB1, ExbD1, HuvB, HuvC, and HuvD (Mouriño et al., 2004). The periplasmic heme-binding protein HuvB delivers periplasmic heme to an inner membrane complex consisting of a permease, HuvC, and an ATPase, HuvD, which subsequently transport heme into the cytosol (Mouriño et al., 2004). Therefore, HuvBCD are required for heme transport, and deletion of either gene results in heme transport deficiencies (Mouriño et al., 2004). HuvZ also plays an import role in heme uptake, and loss of huvZ severely affects the growth of cells when heme serves as the sole iron source (Mouriño et al., 2004). Little is known about the function of HuvZ in heme utilization, and in V. cholerae it is suggested to have a role in heme storage (Wyckoff et al., 2004). HuvX, a predicted intracellular heme delivery protein in V. cholerae (Sekine et al., 2016), is not required for heme uptake in the V. anguillarum 775 plasmidless avirulent strain because deletion of huvX does not cause obvious differences in growth and heme utilization compared with the wild type (Mouriño et al., 2004). However, it is still under debate whether heme utilization is indeed involved in V. anguillarum virulence in nature.

Besides the anguibactin/vanchrobactin and heme uptake systems, four operons encoding putative iron transport systems, including transport of unchelated ferrous (feoABC) and ferric iron (fbpABC1 and fbpABC2), and siderophore ferrichrome transport (fhuABCD), have been identified in the genomes of V. anguillarum strains (Figures 1, 5). The presence of different iron transport systems in V. anguillarum probably results from differences in growth conditions because siderophores can only promote growth under limited iron conditions that must be insufficient for iron uptake under all environmental conditions. In line with this, all the vibrios examined have been shown to have additional iron acquisition systems (Table S3; Payne et al., 2016).

The ferrous iron transporter FeoABC is speculated to be the most ancient iron transport system and widely found among bacterial species including Vibrio species. In V. cholerae, all feoABC genes are required for ferrous iron uptake although their functions have not been fully characterized (Wyckoff et al., 2006; Weaver et al., 2013). However, it is still unknown how ferrous iron passes through the outer membrane for transport by the Feo system in the periplasm. FbpABC, a ferric iron transporter, is also found in vibrios and has been shown to promote better growth at alkaline pH in V. cholerae (Peng et al., 2016). FhuABCD, responsible for siderophore ferrichrome utilization, have been demonstrated to be required for ferrichrome utilization in V. parahaemolyticus and V. cholerae (Rogers et al., 2000; Funahashi et al., 2009).

Notably, although it has been demonstrated that V. anguillarum is capable of using ferric citrate as the only iron source by a siderophore-independent mechanism (Mazoy et al., 1997), we do not find any genes involved in ferric citrate transport in the genome of V. anguillarum or other vibrios. In P. aeruginosa, iron delivered by citrate is suggested to enter the cell as Fe²⁺, and FeoB is further required for citrate-mediated Fe²⁺ uptake (Marshall et al., 2009). Therefore, it is plausible that FeoB of V. anguillarum may also play a role in Fe²⁺ uptake from ferric citrate to maintain iron homeostasis.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.