Xanthomonas citri is the gram-negative bacteria that causes the citrus canker, a disease that infects several species of the citrus genus, specially oranges, still without treatment. The disease is characterized by necrotic lesions on leaves and fruits, which present brown water-soaked margins, culminating with premature fruit drop [1]. The phenotypic symptoms lead to a decrease of the productivity in the citrus production, juice industry, and exportation resulting in massive economical losses for the countries affected, including the main orange exporters such as Brazil, the USA, and China [2].

The mechanism of infection and pathogenicity of the Xanthomonas genus has being largely studied including the functional characterization of genes direct or indirectly related to the capability of the microorganism to spread the disease in plants such as rpf, avr, and hrp [3]. These genes are organized in clusters and their expression affects the virulence, production of secreted proteins, and many proteins localized in the cell envelope, which is related to pathogenicity and evasion from the host immune system defenses [4].

Iron is an essential nutrient normally found as a prosthetic group or cofactor of enzymes involved in several important cellular processes such as nitrogen fixation, amino acid synthesis, citric acid cycle, and DNA biosynthesis [5]. However, the oxidative stress caused by the excess of iron in the cell can be extremely damaging due to the Fenton reaction, in which the reduced form of iron reacts with hydrogen peroxide originating hydroxyl, a compound that can damage proteins and DNA [6]. In order to ensure the organisms’ safety against oxidative stress, the iron must be imported through the cell membranes in controlled amounts by the means of siderophores. These small molecules (200–2000 kDa) are synthesized and secreted by bacteria, fungi, and plants, forming tight and stable complexes with ferric iron. Siderophores are strictly related to the survival of organisms and many times essential for pathogenesis [7].

Despite the relevance of this metal for organisms, there is a lack of information about iron-related genes and their involvement with virulence and pathogenicity of X. citri. In this context, this work presents the putative set of proteins responsible for iron uptake and regulation in X. citri, showing structural and sequential comparisons with orthologs and highlighting the main functional and structural features that might be useful for further studies. The description of the iron components and possible compounds forming complexes for the metal acquisition is relevant for the understanding of X. citri physiology. Moreover, iron-related proteins could be targets for the development of new therapies against the citrus canker.

Iron as a factor of virulence and pathogenicity in different organisms

Given the broad importance of iron for the growth of bacteria, there is no surprise that several animal pathogens have reported siderophore production and internalization as important factor in their virulence and pathogenicity. Examples include pathogenic and uropathogenic Escherichia coli serovars [8] and strains [9], Pseudomonas aeruginosa [10], and Legionella pneumophila [11]. Similarly, siderophores responsible for the iron uptake from the environment are also important for the virulence of the gram positives Staphylococcus aureus [12, 13] and Bacillus anthracis [14] (Table 1).

Table 1 List of pathogens and their relationship with iron and pathogenesis. All pathogens described on the text are followed by the disease they cause or symptoms at the host. Evidences of the relationship between iron and virulence or pathogenesis due to the specific effect of siderophores are shown
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In some phytopathogens such as Agrobacterium tumefaciens [26], Pseudomonas syringae [15], and Ralstonia solanacearum [17], iron import systems were correlated to virulence but not necessarily to pathogenesis.

In Dickeya dadantii, formerly known as Erwinia chrysanthemi, the production of siderophores chrysobactin and achromobactin is highly needed for efficient pathogenesis [18]. Similarly, the necrosis of apple flowers induced by Erwinia amylovora infection is dependent of the ferrioxamine siderophore [19].

Iron and siderophores also have been related to virulence in Xanthomonadaceae (Table 1). In X. campestris pv. campestris, the nutritional dependence of iron and its acquisition was shown to be mediated by cyclic β-(1,2)-glucans in the membrane [27], as well as the import mediated by TonB-dependent receptors [28]. In this bacterium, the transcriptional regulator XibR (Xanthomonas iron-binding regulator) controls the expression of several iron-regulated genes and virulence associated functions [20]. Mutants in genes related to siderophore metabolism in X. oryzae pv. oryzae (colS, XOO1806, acnB, prpR, and prpB) exhibited a deficiency for growth on iron-limiting medium and a decrease in virulence [29].

Proteomic studies of X. translucens pv. undulosa identified many sequences of iron-binding proteins. Further, the analysis of X. translucens proteome for iron-binding proteins revealed that this metal assists on the regulation and function of several secreted proteins linked to its virulence [21]. In X. fragariae, after the disruption of the pyoverdine biosynthetic pathway the bacterium lost its pathogenicity [22].

In X. citri, the genomic analysis aiming the identification of nonribosomal peptides revealed two loci (respectively, locus 1 and locus 2) that showed homology with genes involved with siderophore biosynthesis and lipopeptide synthetases, suggesting a further role of iron in the production of phytotoxins in this bacterium [23]. Iron internalization proteins, including TonB-dependent receptors, were also identified in proteomic analysis [30]. It was suggested that these receptors participate indirectly in biofilm formation and probably have important roles in the adaptability of X. citri by modulating the transport of carbohydrates [24]. A wide genome microarray analysis set up to characterize the pathogenic relevant regulons HrpG and HrpX from X. citri revealed four genes encoding TonB-dependent receptors as part of those regulons (XAC3050, XAC3489, XAC3444, and XAC1143). These genes are mainly known in the transport of iron-siderophore complexes and cobalamin (vitamin B12) into the periplasm [25] (Table 1).

Regulatory mechanisms of iron import in Xanthomonas citri

Despite its importance as a cofactor in the catalysis of several enzymes, high iron concentrations can be deleterious to the cell. The redox activity of iron generating oxidizing molecules and free radicals is extremely damaging to the organism [31]. Thus, the maintenance of the intracellular concentrations of Fe(II) and Fe(III) is critical, and since neither is secreted, the regulation is mainly held by controlling their import [32]. One important regulator of iron uptake is the Fur (Ferric Uptake Regulator) protein, which was first described in Salmonella enterica subsp. enterica ser. Typhimurium [33] and to date is the most known global regulator for iron uptake in several gram-negative microorganisms.

Structurally, the C-terminus of S. Typhimurium Fur binds to Zn(II) on the residues His86, Asp88, Glu107, and His124, which enables the physiological dimer conformation [34,35,36]. The second metal-binding site is formed by the residues His32, Glu80, His89, and Glu100 and binds both Zn(II) and Fe(II). Iron acts as a corepressor in high concentration, allowing Fur to bind to Fur boxes (Fur-binding site or FBS), conserved sequences at the promoter region of key genes related to iron import and siderophore synthesis [20, 37].

The amino acid sequence of XAC1517 protein, the corresponding Fur ortholog in X. citri, was used for searching proteins with structural similarity in the Protein Data Bank. The result revealed a set of metal regulators that shared from 35 up to 58% of amino acid sequence identity, including the P. aeruginosa ortholog (PDB accession code 1MZB) [36] that was used for building the three-dimensional model of X. citri Fur (Table S1, Supplementary material). The amino acid sequence alignment of Fur proteins of E. coli, P. aeruginosa, and X. citri shows the high conservation of the residues that form the two essential metal-binding sites (Fig. 1a, pink and green) and the DNA-binding helix (in yellow). This data can suggest that X. citri Fur has a similar mechanism of transcriptional activation as described in E. coli [32] and P. aeruginosa [36]. The positioning of the residues and the putative DNA-binding helix are presented in the X. citri Fur three-dimensional model (Fig. 1b).

Fig. 1
figure 1

The iron regulatory proteins Fur and XibR identified in Xanthomonas citri. a Amino acid sequence alignment of Fur protein from X. citri (XAC1517) (Xac_Fur), Escherichia coli (b0683) (Eco_Fur), and P. aeruginosa (PA4764) (Pae_Fur). The metal-interacting residues and DNA-binding helix are shown in pink/green and yellow, respectively. b Three-dimensional model of X. citri Fur built from the structural coordinates of the ortholog P. aeruginosa (PDB code 1MZB). The model is colored in the same fashion as the sequential alignment. c Amino acid sequence alignment of X. citri XibR (XAC3733) (xac_XibR), X. campestris XibR (xcb_XibR), and Aquifex aeolicus NTRC4 TF (aae_NtrC). Domains Rec, AAA+, and DNA binding are colored in blue, orange, and green, respectively; X. citri Asp64 conserved in the two other proteins is shown in red bold and the GESGTGK and GAFTGA motifs are evidenced inside the boxes

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Along with Fur, XibR is another transcriptional regulator that was recently characterized in X. campestris [20]. XibR is a transcriptional factor (TF) from the NtrC family, a well-known class of TFs involved in regulation of several processes by binding to alternative σ factors such as σ-54 [38]. It was demonstrated that X. campestris XibR responds to iron depletion and positively regulates motility and chemotaxis, two key processes involved in pathogenesis. Moreover, this protein has a Fur-like function by negatively repressing siderophore synthesis and iron uptake systems [20]. A XibR-like protein with 94% of amino acid sequence identity was identified in X. citri (XAC3733). In addition, according to Blastp X PDB, XAC3733 is structurally similar to the structure of the σ-54 activator NtcC4 from the extremophile Aquifex aeolicus (PDB accession code 3DZD) [39]. The three-dimensional structure of this protein shows three domains: “rec,” with a conserved aspartate residue, the “AAA+” domain that has two motifs for interaction with σ factors (GESGTGK and GAFTGA), and the DNA-binding domain [39]. Based on the amino acid sequence alignment of XAC3733, X. campestris XibR, and A. aeolicus NtrC4 TF, we identified that X. citri XAC3733 conserves the aspartate (Asp64) and the two functional motifs (Fig. 1c, showed respectively, in red and boxes). The high amino acid sequence identity of XAC3733 and X. campestris XibR and the conservation of the functional motifs strongly suggest that it may play a similar function in X. citri. However, despite of the presence and conservation of these proteins, transcriptional regulation involves more important factors, as the cis-elements of each gene belonging to the Fur regulon and also in which processes those genes are involved. In this sense, the understanding of the iron regulation in X. citri is an interesting field to be explored with further functional experiments.

The Xanthomonas citri xss cluster

It is well established that Xanthomonadaceae such as X. campestris [40], X. oryzae, and X. citri [41] share a similar siderophore biosynthesis cluster (named xss, Xanthomonas siderophore synthesis) that is homolog to the operon pvsABCDE of Vibrio parahaemolyticus, responsible for vibrioferrin synthesis [42]. The vibrioferrin-like siderophore produced by Xanthomonadaceae was named xanthoferrin [23]. The xss cluster present in X. citri genome (XAC3175 to XAC3181) and its genome organization is compared with X. oryzae (Fig. 2a). The first gene on X. citri xss is XAC3175 (mphE) and encodes for a 4-hydroxy-2-oxovalerate aldolase that shares 98% and 25% of amino acid sequence identity with mphE from X. oryzae and V. parahaemolyticus, respectively. X. citri mphE is co-transcribed with XAC3176, a TonB-dependent outer membrane receptor (OMR) that shares 44% identity with V. parahaemolyticus ferric vibrioferrin outer membrane receptor PvuA (reference VPA1656) [42].

Fig. 2
figure 2

The xss cluster of Xanthomonas citri. a Gene organization of xss operons localized in X. citri (+) strand and X. oryzae (−) strand. Promoter regions − 10 and − 35 and Fur-binding sites (FBS) are depicted in black and white boxes, respectively. b Amino acid sequence alignment of the plug regions of putative FecA orthologs from X. citri, V. parahaemolyticus in comparison with E. coli FecA plug region. The residues that aligned with E. coli sequence are detached in bold. The numbers correspond to the residues in X. citri sequence

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Since the three-dimensional structures of V. parahaemolyticus and X. citri OMRs are not available, we used the known sequence of E. coli FecA OMR to compare the residues in the plug region. In FecA protein, this region contains the key residues that give specificity and perform interactions with the correspondent siderophore: Thr138, Arg155, Gln176, and Ser180 [43]. The amino acid sequence alignment of E. coli FecA, X. citri XAC3176, and V. parahaemolyticus PvuA shows that the plug region of XAC3176 conserves three from the four residues that in E. coli are involved in the ligand coordination, Thr106, Gln141, and Ser145 (Fig. 2b). The FecA Arg155 is replaced by a serine in XAC3176 (Ser120) and PvuA. The complete alignment of the proteins XAC3176, PvuA, and FecA is presented in Figure S1 (Supplementary material). The similar genome organization, the putative role of the enzymes, and the conservation of some key residues from the plug region suggest that XAC3176 might be the xanthoferrin outer membrane receptor in X. citri. Indeed, more robust experimental data should be performed in order to understand what is the transport function that XAC3176 plays in X. citri.

The next operon has 5 genes that encode enzymes involved in siderophore biosynthesis (XAC3177, XAC3178/XssB, XAC3180/IucA, and XAC3181/LysA) and a member of the major facilitator superfamily protein (XAC3179/YceE) (Fig. 2a). A list of all proteins of X. citri xss and their correspondent orthologs in X. oryzae, V. parahaemolyticus, and E. coli, including amino acid sequence identities, is presented in Table S2 (Supplementary material).

The FeoABC of X. citri: The ferrous iron import system

Due to iron importance for the cell homeostasis, it is not unusual to find more than one route for its import. Despite the well-known systems of metal chelating siderophore internalization, iron can also be internalized in its ferrous form, Fe(II), after reduction of the siderophore-Fe complex in the periplasm or even outside the cell by specific reductases. In the latter case, the ferrous iron crosses the outer membrane through porins [44]. The Feo system (Ferrous iron transport) is the main pathway by which ferrous iron crosses the inner membrane of gram-negative bacteria. It was first described in E. coli [45] and regulated by Fur in a similar fashion as the siderophore producing and other related genes [46].

Genetically, the E. coli Feo system is encoded by feoABC operon consisting of three co-transcribed genes that encode two small proteins for signaling (b3408 and b3410) and the permease for ferrous iron transport (XAC3409). Several studies have shown a correlation between those genes and virulence in different animal pathogens such as Helicobacter pylori [47], Pseudomonas gingivalis [48], Clostridium jejuni [49], and S. typhimurium [50]. Similarly, the feoABC operon of X. oryzae pv. oryzae (XOO2900 to XOO2898) was related to virulence in planta [41] and its ortholog is present in X. citri (XAC1854-XAC1856), including the Fur-binding site (Fig. 3a).

Fig. 3
figure 3

The feoABC operon of Xanthomonas citri. a Gene organization of the feoABC operon in X. citri. Promoter regions − 10 and − 35 (black box) are showed as well as the Fur-binding site (FBS). b Comparison between X. citri and E. coli FeoB proteins. (I) Three-dimensional structure of E. coli FeoB N-terminal domain (PDB 5FH9) is shown in cartoon representation (helices in red and beta strands in yellow) and highlights the functional G5 motif localized in a coil region (green spheres). A comparison of E. coli and X. citri G5 regions is shown. (II) Transmembrane prediction of X. citri FeoB using the TMHMM program (http://www.cbs.dtu.dk/services/TMHMM/) shows a cytoplasmic N-terminal domain (blue line) and 9 alpha-helices (in red), similar to what is described for E. coli FeoB. c Sequential alignment of FeoC from X. citri (XAC1856) (Xac_FeoC), X. oryzae (XOO2898) (Xoo_FeoC), and E. coli (b3410) (Eco_FeoC), the putative functional cysteine residues are depicted in red bold

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In E. coli, the main component of feoABC operon is FeoB, likely the permease that allows the passage of ferrous iron from the periplasm to the cytoplasm [51]. FeoB contains two domains, a cytoplasmic N-terminal domain that shows a GTPase activity, and a C-terminal domain that consists of transmembrane helices. The three-dimensional structure of E. coli FeoB N-terminal domain (PDB code 5FH9) [52] is showed in Fig. 3b-I. The structure is presented in cartoon red (helices) and yellow (beta strands) and the G5 motif that provides hydrophobic and electrostatic interactions with GTP is shown in green spheres [53]. This motif is located in a coil region, consisting of seven residues conserved when compared with X. citri FeoB (Fig. 3b-I). The C-terminus of FeoB consists of 8 α-helices that span the cytoplasmic membrane with two gate motifs, each with 4 stretches of residues and a conserved cysteine that is important for metal binding [51, 54]. Similar to the E. coli protein, the secondary structure and transmembrane helices prediction of X. citri FeoB (XAC1855) revealed a cytoplasmic N-terminal (residues 1–200) that shares 40% of amino acid sequence identity with E. coli ortholog, and 9 alpha-helices forming a transmembrane C-terminal (Fig. 3b-II).

E. coli FeoA (b3408), with orthologs in X. oryzae (XOO2897) and X. citri (XAC1854), is predicted to be a membrane-interacting protein that works along with FeoB [51]. E. coli FeoC, a putative TF, is a highly disordered protein containing a large loop region on its C-terminus with four conserved cysteine residues forming the binding site for Fe(II). Similarly to the Fur protein system, Fe(II) is speculated to act as a corepressor in FeoC [51]. Based on the primary sequence of FeoC from E. coli (Eco_b3410), an alignment was performed with X. oryzae (XOO2898) and X. citri (XAC1856) putative FeoC (Fig. 3c). X. citri FeoC shares 89% of sequence identity with X. oryzae protein, but only 13% with the putative ortholog of E. coli. Despite the low identity, both proteins conserve the important functional cysteines present in E. coli protein. The importance of FeoC along with FeoB and FeoA for X. citri pathogenesis remains to be investigated.

Putative OMRs and ATP-binding cassette transporters involved in siderophore-Fe uptake in X. citri

Siderophores of different classes can bind Fe(III) in the extracellular domain and be internalized by specific OMRs of gram-negative bacteria. In E. coli, four OMRs are well characterized: the ferrichrome transporter FhuA (b0150) [55], ferrienterobactin transporter FepA (b0584) [56], ferric citrate transporter and signal transducer FecA (b4291) [43], and the cobalamin transporter BtuB (b3966) [57]. Structurally, those proteins share a high conserved folding consisting of two well defined domains: a N-terminal domain (plug domain), which is mainly responsible for the interaction with the siderophore and that mediates its internalization via conformational changes, and a C-terminal consisting of a β-barrel structure that forms the pore where the plug domain is located [58,59,60]. As previously suggested [61, 62], the rearrangement of the plug domain allows the passage of the siderophore from the extracellular medium to the periplasm [37]. It has been demonstrated that the binding of the antibiotic rifamycin to E. coli FhuA culminated in several allosteric transitions including the helix-to-coil transition in the called N-terminal switch helix [63]. This helix harbors the TonB box motif, responsible for recruiting and binding of TonB, which is important for the energization of the system.

TonB works by mediating the coupling of the chemiosmotic gradient from the inner to the outer membrane transport with the assistance of two proteins: ExbB and ExbD. Although the mode of action of the three proteins together as a complex is not yet fully understood, they compose an apparatus that cooperatively energizes the OMR and mediates the structural rearrangement that will culminate with the siderophore internalization to the periplasmic environment [58, 64, 65].

To search the presence of putative iron-related OMRs orthologs in X. citri, amino acid sequences of the classical FhuA, FepA, FecA, and BtuB of E. coli were used in Blastp [66] against the X. citri databank. Respectively, four X. citri sequences were obtained with query coverage higher than 70% and amino acid sequence identity higher than 30%: FhuA ferrichrome-iron receptor (XAC2185), PfeA siderophore receptor (XAC3620), FecA citrate–dependent iron transporter (XAC3176), and BtuB outer membrane receptor for transport of cobalamin (XAC3194) (Table 2, outer membrane receptors). To complement the analysis, the identified amino acid sequences were used for Blastp against the Protein Data Bank. Besides the abovementioned E. coli proteins, the search revealed that X. citri XAC2185 (FhuA) sequence has structural similarities with the OMRs of pyoverdine FpvA (PDB 1XKH) and pyochelin FptA (PDB 1XKW) from P. aeruginosa and the ferric enterobactin OMR PiuA (PDB 5FP1) from Acinetobacter baumannii (Table S3, Supplementary material).

Table 2 Putative orthologues of the iron uptake systems from Escherichia coli identified in Xanthomonas citri. (−) not identified or (nr) amino acid sequence identity with coverage and similarity lower to 70% and 30%, respectively
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The sequences of all putative orthologs were used for structural alignment and, when available, for identification of residues involved with siderophores coordination. The gene cluster and operon organization of the identified orthologs in X. citri were compared with E. coli (Fig. 4 and Table S4, Supplementary material). Residues that coordinate the ferrichrome in E. coli FhuA (PDB 1BY5) and pyoverdine in P. aeruginosa FpvA [10, 67] were identified and compared with X. citri FhuA sequence showing no sequential similarity (Fig. 4a). Genomewise, E. coli fhuA is followed by a co-transcribed operon for the ATP-binding cassette (ABC) transporter fhuBCD, which encodes a full importer consisting of permease, ATPase, and a substrate-binding protein, respectively, that are responsible for internalizing ferrichrome through the inner membrane [68]. However, in X. citri genome, fhuA is located in the same operon as two hypothetic genes (XAC2184 and XAC2186) not being co-transcribed with components of an ABC transporter system like evidenced in E. coli genome (Fig. 4a) (Table 2).

Fig. 4
figure 4

Partial amino acid sequence alignment and cluster organization of E. coli OMRs and their orthologs in X. citri. The percentage of amino acid sequence identity between X. citri proteins and orthologs is shown. Gene references, names, and colors follow the KEGG code. Residues involved in the coordination of the ligands in the available structures are presented in bold. The gene cluster organization in X. citri is also compared with E. coli. a Partial alignment of E. coli FhuA bound to ferrichrome (PDB 1BY5) and its orthologs in X. citri (XAC2185) and P. aeruginosa FpvA bound to pyoverdine (PDB 1XKH). The localization of fhuA gene organization in X. citri is compared with E. coli. b E. coli FepA (PDB 1BY5) and its orthologs in X. citri (PfeA, XAC3620) and P. aeruginosa (PfeA) bound to azotochelin, protochelin, and enterobactin (PDB references 5NR2, 5NC4, and 6Q5E, respectively). c Amino acid sequence alignment of E. coli FecA and its ortholog in X. citri. Residues that interact with ferric citrate in E. coli protein (PDB 1KMP) are shown in bold. The xss cluster is compared in the two strains. d E. coli BtuB amino acid sequence and residues that interact with cobalamin (PDB 1NQH, in bold) aligned with the amino acid sequence of the X. citri ortholog

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The comparison of X. citri PfeA with the E. coli FepA (47% identity) and P. aeruginosa PfeA (50% identity) showed that residues involved in binding of azotochelin, protochelin, and enterobactin in the P. aeruginosa protein are present in X. citri PfeA, suggesting a possible interaction with these or similar siderophores that could be experimentally investigated. As it was shown for fhuA, in the gene cluster to which pfeA belongs, there are no genes encoding the corresponding ABC transporter components (Fig. 4b).

X. citri FecA, the OMR found as part of xss, presented five from seven residues involved in the ferric citrate binding when compared with E. coli FecA (PDB 1KMO) (30% identity). E. coli FecA–binding site is composed mainly of positively charged residues to interact with negatively charged ferric citrate, and the sequential similarity of X. citri FecA might indicate that it could import a similar siderophore. Still, the higher identity with the vibrioferrin receptor from V. parahaemolyticus (44%) that has no structure resolved and the presence of fecA in xss cluster strongly suggests that xanthoferrin is imported by this OMR, not citrate.

E. coli fecA is preceded genomically by a σ-19 factor (fecI) and an anti-σ factor (fecR), which is an already described alternative regulation system along with Fur [69] and followed by the complete ABC transporter for dicitrate import fecBCDE. In X. citri fecA gene cluster, at least three genes named bfeA encoding putative outer membrane receptors for enterobactin (XAC3166 to XAC3168) are found upstream from the xss operons, but again, no ABC transport genes were identified (Fig. 4c, Table 2).

Regarding the cobalamin import, in X. citri more than one ortholog of BtuB were identified but only XAC3194 showed 34% of amino acid sequence identity with the E. coli putative ortholog. Still, the comparison with the classical BtuB from E. coli did not give any clues that it could be functional for the vitamin binding. On the opposite, an operon dedicated to cobalamin synthesis and assimilation, including the transcriptional regulator btuR (XAC3191 to XAC3184), is located just downstream of the btuB gene (XAC3194) (Fig. 4d).

The identification of the four putative OMRs orthologs in X. citri indicates that this bacterium has distinct systems for uptake of siderophores, including some very interesting candidates for experimental approaches such as PfeA (XAC3620) and FhuA (XAC2185). Interestingly, no corresponding ABC transporters were identified at least in the same operons or clusters that encode the OMR genes. This fact suggests that the mechanisms for siderophore uptake and iron assimilation in X. citri need to be further explored.

Iron-related genes in different proteomics analyses of X. citri

Key proteomic analyses have been performed in the past 5 years in order to access X. citri protein profile in different growing conditions. The first analysis was based on the comparison of the proteins that were expressed during infection of X. citri in Citrus sinensis leaves (in planta) and in nutrient broth (NB) rich media (in vitro) [70] (Table 3). The effect of the phosphate in X. citri proteome was also evaluated in A medium, known as the Pho regulon inducer, and A medium added of inorganic phosphate (Pi) [71]. We observed that significant amount of proteins identified in these proteomes belong to the cellular envelope such as OMRs that were up- or down-regulated. Different orthologs of the E. coli cobalamin transporter BtuB (XAC3050, XAC3444, and XAC2531), ferric enterobactin receptors (XAC3166, XAC3168, XAC3207), and citrate/ferrin transporters (XAC3334, XAC4368, XAC3176 XAC1023) were regulated by phosphate levels and during plant infection. X. citri XAC0823 (PhuR), another putative ortholog of E. coli FecA (b0150), is present in phosphate depletion in A medium and it is down-regulated in presence of Pi. Besides the membrane receptors and transporters, Fur regulator (XAC1517) and proteins from xss cluster (XssA and XssB) were up-regulated in Citrus sinensis leaves and A medium + Pi showing the importance of xanthoferrin biosynthesis during infection and stress conditions [70, 71].

Table 3 Iron-related proteins identified in different experimental proteomic studies of Xanthomonas citri. Conditions analyzed phosphate levels [71], rich medium and infection conditions [70], and biofilm [24]. A medium: Tris 120 mM, NaCl 80 mM, KCl 20 mM, Na4Cl 20 mM, Na2SO4 3 mM, MgCl2 1 mM, ZnCl2 51 μM, bacto peptone 0.5%, glucose 0.5%; CaCl2 0.2 mM; A medium + Pi (10 mM NH2PO4); NB, nutrient rich broth; Citrus sinensis (sweet orange) leaves. The name of the proteins is followed by the KEGG reference in parenthesis. Some proteins were identified in more than one reference
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TonB receptors also were identified in biofilm proteome when compared with planktonic cells but none of those described in this work [24] (Table 3). Many of the proteins are described with similar functions indicating that X. citri has redundancy of systems for iron acquisition during X. citri growth, infection, and pathogenesis, which detaches the relevance of the element for the bacterium.

Discussion

Iron assimilation system in X. citri, as in many organisms, is a complex network of transcription regulators, siderophore synthesizing proteins, efflux pumps, and inner and outer membrane proteins that together promote the fine homeostasis needed for maintenance of the micronutrients in appropriate levels inside the cell. Despite some similarities, specially regarding the regulators, most of the systems identified in X. citri lack the complementary proteins for full import of the siderophores, such as known for E. coli, or showed low identity with the most studied proteins in other microorganisms. In the literature, it is not unusual that a great number of siderophore importers are found in organisms that do not possess the machinery for their synthesis or export; those are commonly known as xenosiderophores. Examples of siderophores produced by one species but used by another are described in the literature [72] and represent an adaptative advantage found in several gram-negative bacteria when competing with other organisms for scarce sources of metal [37].

Considering the ability of xenosiderophores import and the putative OMRs presented in this study, it is plausible that X. citri can transport siderophores that it does not produce. That would also explain the absence of Fur boxes in the promoter regions of many genes described in this work. On the contrast, the presence of a Fur box at the promoter of the xss OMR XAC3176 strengthens the hypothesis that this protein is responsible for the import of a X. citri siderophore xanthoferrin, the homolog to X. oryzae siderophore produced by its own xss [41]. Indeed, XAC3176, XAC3177, and XAC3178, all belonging to the same operon that has Fur-binding site, were identified in all proteomic analyses corroborating their relevance for the bacterium.

As for the inner membrane protein FeoB (XAC1855), sequential- and structural-based analyses suggest that the ferrous form of iron could be internalized as described in other species. Besides the OMR XAC3176, most likely the xanthoferrin importer in X. citri, other proteins from xss cluster that are localized in the cytoplasm were also identified along with the inner membrane exporter XAC3179. Further analyses on the structure and function of each individual protein in xss might help in addressing the production of xanthoferrin in X. citri.

Finally, the two putative transcriptional factors Fur and XibR from X. citri were recognized in the bacterium genome and showed high sequential identity to the extensively characterized P. aeruginosa Fur. On the other hand, X. citri FeoC is closely related to its ortholog in X. oryzae, but poorly comparable with the well-characterized E. coli FeoC. Based on the set of results, all the proteins described in this study and those evidenced in proteomics analyses were organized along with their proposed cellular localization (Fig. 5).

Fig. 5
figure 5

Schematic localization of the Xanthomonas citri iron-related proteins that were previously described in the literature and the ones identified in this work (evidenced with an asterisk). Proteins were separated according to the defined categories of KEGG database

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Altogether, the findings of this study that were based on the compilation of bioinformatics data and comparisons with proteins described in the literature give functional insights to the X. citri pathways for iron internalization and assimilation. Further studies are still needed to address the relationship among the many proteins described and their function in iron uptake, virulence, and pathogenesis of X. citri. Moreover, these proteins might become suitable as targets for development of effective growth inhibitors against this phytopathogen in the future.