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A genome-wide analysis of desferrioxamine mediated iron uptake in Erwinia spp. reveals genes exclusive of the Rosaceae infecting strains

Erwinia amylovora is the etiological agent of fire blight, a devastating disease which is a global threat to commercial apple and pear production. The Erwinia genus includes a wide range of different species belonging to plant pathogens, epiphytes and even opportunistic human pathogens. The aim of the present study is to understand, within the Erwinia genus, the genetic differences between phytopathogenic strains and those strains not reported to be phytopathogenic. The genes related to the hydroxamate siderophores iron uptake have been considered due to their potential druggability. In E. amylovora siderophore-mediated iron acquisition plays a relevant role in the progression of Fire blight. Here we analyzed the taxonomic relations within Erwinia genus and the relevance of the genes related to the siderophore-mediated iron uptake pathway. The results of this study highlight the presence of a well-defined sub-group of Rosaceae infecting species taxonomically and genetically related with a high number of conserved core genes. The analysis of the complete ferrioxamine transport system has led to the identification of two genes exclusively present in the Rosaceae infecting strains.

Fire blight is one of the major threat to Rosaceae with a potentially disastrous economic impact on apple and pear production 1 . With favorable environmental conditions, an outbreak can cause the loss of entire annual harvests. The etiological agent of the disease is Erwinia amylovora, one of the top ten known plant pathogens 2 . The Erwinia genus comprises species that are plant pathogens, non-pathogen, epiphytes, and even opportunistic human pathogens [3][4][5] . E. amylovora can infect a wide range of hosts, comprehensive of apple, pear, hawthorn, cotoneaster, rubus, etc 6 . Earlier comparative genomic studies, focused on virulence genes, suggest that E. amylovora host specificity and pathogenicity is driven by the absence or presence of certain genes 7 .
In living organisms, iron is a fundamental element, present as a cofactor in proteins and enzymes (e.g., iron-sulfur clusters, heme groups, etc.). A broad spectrum of biological relevant reactions are catalyzed by the reversible Fe(II)/Fe(III) redox pair 8 . The restriction on the availability of iron led to the development of highly selective systems for its acquisition, either directly (e.g. through transferrin, heme or hemoproteins) or indirectly, through hemophores or siderophores. In particular, siderophores are small molecules (usually < 1 kDa) that complex ferric iron and are among the strongest Fe(III) chelators 9 . Iron uptake is one of the main molecular pathways involved in the fire blight disease progression 10,11 . Not all Erwinia species are able to synthetize their own siderophores, suggesting that iron uptake is a potential niche adaptation factor 12 . E. amylovora mutants, with defective siderophore biosynthesis and uptake, showed an important reduction in their growth on apple flowers compared to the wild-type strain 13 . In E. amylovora, siderophore mediated iron uptake is dependent on desferrioxamines (DFOs) 14 : molecules consisting of alternating diamine and dicarboxylic acid building blocks 1 Bioorganic Chemistry and Bio-Crystallography laboratory (B2Cl), Faculty of Science and Technology, Free University of Bolzano, Piazza Università 5, 39100, Bolzano, Italy. 2  linked by amide bonds. The major product of DFOs biosynthesis in E. amylovora is nocardamine (desferrioxamine E, DFO-E). Three proteins, namely DfoJ, DfoA and DfoC, are responsible for the biosynthesis of DFO-E starting from lysine 15,16 , these proteins are encoded by a single gene cluster, the dfoJAC operon. Together with the DFOs biosynthetic pathway proteins, the bacterium expresses a specific membrane receptor, called FoxR, necessary for the transport of ferrioxamine complexes across the outer membrane 17,18 . Transport across the bacterium inner membrane, possibly depends on the periplasmic binding protein-dependent ABC transporter complex FhuABCD, as reported for other hydroxamate siderophores of enteric bacteria e.g. in Escherichia coli [19][20][21] and Salmonella enterica 22 . Ferrisiderophore complexes are very stable, and the mechanism of iron release can occur with three different mechanisms: hydrolysis of the siderophore, proton-assisted dissociation of the complex, and reduction of the metal center 23 . The lack of specific hydrolases in E. amylovora and the incompatibility of the cytoplasmic pH with the proton-assisted dissociation suggests that the iron is released after its reduction (ferrioxiamine has a lower affinity for Fe(II) than for Fe(III)), as in E. coli 24 .
The aim of the present study is to understand the genetic differences within Erwinia spp. between the species reported to be phytopathogenic and the one never reported to be correlated to plant diseases (here considered as non-phytopathogenic). A special focus is dedicated at the genes related to the hydroxamate sidephores iron uptake. We used a comparative genomic approach and selected a specific dataset of Erwinia spp. covering 11 genomes from phytophatogenic species, and 8 genomes from non-pathogenic. The genome selection has been limited to the available ones completed and correctly annotated. Average Nucleotide Identity (ANI), phylogenetic inference based on conserved marker genes, pangenomic analysis and molecular diversity analysis have been performed to get insights about the relevance of siderophore mediated iron acquisition in the evolution of pathogens' hosts selectivity and virulence.

Results
In order to work on a balanced dataset of the Erwinia genus, 11 plant pathogens and 8 non-pathogens genomes were selected among the sequenced genomes in Erwinia ( Average Nucleotides Identity and phylogenetic analysis. The ANI values and the phylogenetic analysis consistently highlighted a clear division between the Rosaceae infecting pathogens (RIP) and the other species (Non Rosaceae infecting pathogens, NRIP). The genomes of the RIP group show higher similarity, with pairwise ANI values always higher than the ones from the other species and a large portion of genome aligned (above 6Mbp). Other species instead, had lower pairwise ANI values, also when the alignment lengths was comparable to the ones in the pathogenic species (Fig. 1a). Furthermore, the PhyloPhAn tree (Fig. 1b) reports a shorter phylogenetic distance between RIP species compared to the NRIP species. The tree topology is slightly different from the one in Fig. 1a, however the large basal split between RIP and NRIP is conserved. Erwinia tasmaniensis is an exception: in fact, although it has never been reported to be a pathogen, it shows a high genome similarity with the RIP group.
Core and accessory genome in Erwinia. Clusters of orthologous proteins were created using Anvi' o, a binary matrix including the presence of genes in the different genomes was exported to perfom a pangenomic study. The core genome shared between RIP and NRIP is plotted in the Fig. 2a. A total of 1551 genes (58.9% of the core genes) are shared between the two groups (RIP and NRIP, hard-core genes), the number of soft-core genes (i.e. core within one of the two groups, but not in the other), is 1034 (39.3%) and 47 (1.8%) in RIP and NRIP, respectively. The multivariate analysis plot (Fig. 2b) displays patterns that are coherent with the findings of our phylogenetic analysis. All the RIP and E. tasmaniensis are concentrated in a closer space forming a narrow cluster in the center of the plot. The other species are scattered in both directions along the NMDS1 and NMDS2 axes within the plot without forming any distinguishable cluster.
presence/absence of genes related to hydroxamate siderophores iron uptake and polymorphism. Table 2 summarizes the presence/absence of coding sequences (CDS) for genes related to hydroxamate siderophores iron uptake in the 19 Erwinia genomes taken into account. As shown in Table 2, 9 genes have been considered as marker. All the marker CDS have been found in the RIP and in E. tasmaniensis; in NRIP species the number of the CDS (found with a complete coding sequence) of interest ranged from 2 to 4 ( Table 2). The most prevalent gene is fhuB (coding part of the hydroxamate import system), found in 15 out of 19 strains, followed by dfoC, dfoA and foxR (synthesis and transport of ferrioxamine). The least present are fhuD (periplasmic binding protein) and sidE (siderophores utilization protein), both found only in RIP and E. tasmaniensis. The combination of the PhyloPhlAn analysis and the CDS presence is represented in Fig. 3. A monophyletic clade constituted by all the RIP and E. tasmaniensis contains all the CDS screened. Moreover, the CDS for fhuD (in orange) and sidE (in red) can be found only in this clade. The CDS for fhuB (in pink) is almost ubiquitous but it is absent in E. gerundensis, in one of the E. iniecta (E. iniecta B149) and in both E. billingiae. dfoA. A complete CDS for the gene dfoA was found in all RIP, in E. tasmaniensis, both strain of E. iniecta, E. toletana and one of the two E. billingiae strains (EB661). The other E. billingiae strain (OSU 191) missed an in-frame start codon. From none of the remaining species was possible to retrieve the homologous gene neither through blast, nor through the annotated genbank file. In RIP, the gene is conserved, with a Pi value below 0.1, whereas in NRIP there is a highly variable region around 400 bp. Despite the lower conserved of dfoA gene in NRIP, there are two noticeable conserved regions around 200 bp and 1100 bp (Fig. 4a).
www.nature.com/scientificreports www.nature.com/scientificreports/ dfoC. A complete CDS for dfoC was found in all the RIP, in E. tasmaniensis, in both E. billingiae and E. iniecta, in E. olea and E. mallotivora. It was not possible to retrieve the CDS from the annotated file. A remarkable feature of this alignment was a large (12 nt), in-frame insertion found in the two E. iniecta strains and E. toletana. In RIP, the gene is very conserved and in particular the region around 400 bp and the last 500 nucleotides. In the NRIP, the gene is generally less conserved than in RIP, with conserved 3 areas (regions around 400 bp, 1300 bp and 2000 bp, Fig. 4b). The variability pattern in this gene show a peak of variation at 1200 bp in NRIP that was not present in RIP.

dfoJ.
A complete CDS was found using blast for all E. amylovora, the two E. pyrifoliae, Erwinia sp. Ejp617, E. pyriflorinigrans and E. tasmaniensis. It should be noted that from the genbank file, a feature annotated as dfoJ was present only in one out of three E. amylovora genomes (CFBP1430) and one E. pyrifoliae (DSM12163). A sequence with high identity with the dfoJ gene was also found in E. oleae, however, this gene was lacking an in-frame stop codon, even after manual inspection of the 5′ downstream region. The dfoJ gene was found only in RIP, and it display a relatively low value of Pi all along the sequence compared to the other genes (Fig. 4c).
fhuA. This gene was annotated only in two out of three E. amylovora strains (ATCC49946 and CFBP1430), and in the two E. pyrifoliae. However, a complete coding sequence was found in all E. amylovora and E. pyrifoliae, and in Erwinia sp., E. tasmaniensis, E. persicina, E. piriflorinigrans, both E. billingiae and E. gerundensis. The latter featured a TGA (opal) stop codon, whereas all other species have a TAA (ochre). As for the previous genes, the polymorphism is higher in NRIP than in RIP (Fig. 4d). This gene had a peak of variation in RIP in correspondence of a conserved region of NRIP, at 400 bp.
fhuB. Similar to fhuA, this gene was annotated in the same genomes, but found through blast in 16 out of 19 genomes (leaving out only the two E. billingiae and E. gerundensis). For E. iniecta B149, however, it was not possible to retrieve the whole species due to the fragmentation of the assembly (the 5′ end was cut by a stretch of "N"). Therefore, for the subsequent analysis, fhuB sequences for this genome was removed. The differences in the degree of polymorphism between the two populations (RIP vs NRIP) is evident (Fig. 4e), with maximum in the regions around 900 bp and 1500 bp.
fhuC. This was detected in all rosaceae infecting strains, in E. tasmaniensis and E. gerundensis (that once more had a TGA stop codon, and the most diverging sequence), although it was annotated only in the same four strains as fhuA and fhuB. The gene is conserved in RIP, with slightly higher polymorphism in the last 150 nucleotides (Fig. 4f).
fhuD. This was detected in all Rosaceae-infecting strains and in E. tasmaniensis, although it was annotated only in the same four strains as fhuA and fhuB. The gene is conserved in RIP (Fig. 4g). www.nature.com/scientificreports www.nature.com/scientificreports/ foxR. This gene was detected in all RIP plus the two E. billingiae, the two E. tracheiphila, E. oleae, E. tasmaniensis. It was annotated in the genbank files of the two E. pyrifoliae and E. amylovora CFBP1430. The gene polymorphism is higher in NRIP than in RIP (Fig. 4h). In RIP, the most conserved regions are around 400 bp, 750 bp and 1300 bp, while in NRIP the most conserved regions are at 300 bp and 1600 bp. Once again, a peak of nucleotide variation in NRIP was detected in correspondence of a conserved region in RIP, at 1300 bp.
sidE. This gene was detected in all Rosaceae-infecting strains, plus E.tasmaniensis. In RIP, the gene is overall conserved, with a small peak at 700 bp (Fig. 4i).

Discussion
The Erwinia genus includes phytopathogens affecting Rosaceae relevant in fruit production (e.g., Malus sp. and Pyrus sp.). In E. amylovora, the ferrioxamine biosynthesis and transport has been reported to be involved in the progress of the Fire blight disease 10-12,15,16 which makes it a worth studying target pathway. In this work, a specific dataset of Erwinia spp. has been analyzed comparing at first the whole genome, then a group of conserved proteins, and at last focusing on specific siderophore genes. The ANI (Fig. 1a), the phylogenetic analysis (Fig. 1b), the pangenome and multivariate analysis based on the presence/absence of all genes (Fig. 2b) are coherent. The whole analysis highlights that a defined group of species is present in the heterogeneous Erwinia genus. All the species in this group, with the exception of E. tasmaniensis 25 , have been reported to be Rosaceae infecting pathogens [25][26][27][28][29] . Our results support that the lack of only few specific genes (involved in the biosynthesis and regulation of harpins, effectors and amylovoran) hints for a possible non-pathogenicity of E. tasmaniensis ET1/99 as suggested by Borruso et al. 7 and others previously [30][31][32] . Erwinia spp. Ejp617 in all analysis clusters very close to the group of E. pyrifoliae although not as ingroup. According to our results Erwinia spp. Ejp617 isolated in Japan 28 could be assigned within the species E. pyrifoliae as has been done in other reports 28,33 . All phylogenetic analysis (Figs 1 and 2b) suggest a different evolutionary rate within the RIP. These difference can be appreciated by looking at the branch length in Fig. 1b: the distance between E. amylovora, E. pyrifoliae, Erwinia sp Ejp617, E. piriflorinigrans and E. tasmaniensis is shorter than any distances among other species. The number of soft-core genes in the two groups is very different (Fig. 2a), suggesting a high level of host-adaptation required in the RIP for infecting the plant. The genomic similarity of RIP is revealed by the selected marker genes. The high level www.nature.com/scientificreports www.nature.com/scientificreports/ of host-adaptation is reflected in the iron uptake system, where the Pi value/polymorphism for all the genes from NRIP species is higher than in RIP (Fig. 4). Moreover, there are some contrasting nucleotide positions (e.g. 1200 bp in dfoC and 400 bp in fhuA) where there is a maximum of Pi value for one population and minimum for the other one. These regions could play a key role in the enzyme function and/or be part of the active site. Complete CDS for ferroxiamine siderophore biosynthesis (dfoJAC), outer membrane receptors (fhuA and foxR), periplasmic binding protein (fhuD), ABC cassette type receptor components (fhuB and fhuC) and siderophore utilization protein (sidE) are always present in all the Rosaceae infecting Erwinia (plus E. tasmaniensis). The role of ferrioxamine synthesis and foxR receptor in the disease has been previously reported 13,14,17,18 and accordingly to our results, the complete transport system is relevant as well. Moreover, while some genes of the biosynthesis and the receptors are shared between RIP and NRIP, the genes coding for the periplasmic binding protein (fhuD) and the siderophore utilization protein (sidE) are strictly conserved in the RIP (Fig. 3). According to the PhyloPhlAn analysis, the epiphytic bacteria E. billingiae is the farthest Erwinia in respect of the RIP. However, it shares both the outer membrane receptors for hydroxamate siderophore and 2 out of 3 enzyme required for the ferrioxamine biosynthesis. E. billingiae tends to invade plants necrotic tissue 34,35 . E. billingiae could withdraw the intermediates and the ferrioxamine from the RIP, slowing down its growth, while scavenging the iron available in the plant tissues necrotized by pathogens. This behavior suggests a possible symbiosis/antagonism with the RIP 36,37 even in the synthesis of siderophores. The absence of the almost ubiquitous gene fhuB from E. billingiae, E. gerundensis, and E. iniecta B149 could be explained with two hypotheses: i) it is possible that the gene, present in the ancestor, has been lost by the two derived species; or ii) a fragmented assembly may result in a missing gene. The latter is the most probable for E. iniecta B149, because the assembly consists of 121 contigs, whereas E. gerudensis consists of 3 contigs. However, in both E. billingiae and in E. gerundensis, the gene was most likely lost. www.nature.com/scientificreports www.nature.com/scientificreports/

Methods
In order to have a balanced dataset of the Erwinia genus (11 plant pathogens vs 8 non-pathogens) genomes to be screened were selected among the sequenced genomes in Erwinia according to the list in Borruso et al. 7 , both fasta and genbank format were downloaded from genbank on the 14th of February 2018. To avoid biases due to the much larger number of sequenced E. amylovora genomes we selected the three genomes with the highest completion score, namely E. amylovora strain CFBP1430, strain 49946 and strain E-2. Eventually, 19 genomes, spanning 13 species, were selected. Seven genomes (in four species), belonged to species known Organisms fhuB dfoC dfoA foxR fhuA dfoJ fhuC fhuD sidE   www.nature.com/scientificreports www.nature.com/scientificreports/ to infect Rosaceae, the remaining 12, spanned 9 species in which this phenotype is not reported (Table 1). Siderophore mediated iron uptake genes of Erwinia spp. were selected for their relevance: dfoA (ordered locus name EAMY_3239), dfoC (EAMY_3240), dfoJ (EAMY_3238), fhuA (annotated as 3 fragments: EAMY_2775, EAMY_2776 and EAMY_2777), fhuB (EAMY_2772), fhuC (EAMY_2774), fhuD (EAMY_2773), foxR (EAMY_3241), sidE (EAMY_3562). The gene sequences from E. amylovora CFBP1430 have been used as reference. A first genome-wide comparison was done using the Average Nucleotide Identity (ANI), using the software PYANI 38 , and the output was elaborated using the software DiMHepy (https://github.com/lucaTriboli/ DiMHepy). A phylogenetic tree was built on the concatenation of 400 conserved proteins using PhyloPhlAn 39 , and the tree was drawn and annotated (e.g. presence absence of genes was reported on each leaf of the tree) using IToL 40 . To infer the number of shared protein clusters, genomes were imported in Anvi' o 41 , the pipeline followed the standard pangenomic workflow with all default parameters. A binary matrix containing all protein clusters of the different genomes was exported and analyzed via Non Multivariate Analysis (NMDS) based on Euclidean distance. Venn diagram was generated to display the number of core genes. Briefly, we considered as core genome the pool of genes present in all genomes of RIP and NRIP separately. Coding sequences (CDS) for each of the 9 genes were searched through BLASTn 42 using as query the CDS of E. amylovora CFBP1430. When partial or no CDS were detected, alignments were manually curated by retrieval of the homologous flanking sequences from the fasta files. DNA polymorphism for each selected gene has been analyzed using the sliding window method with the software DnaSP v6 43 and expressed as Nucleotide diversity (Pi) 44, 45 . The parameters for the analysis have been established in function of the gene length. The window length and the step size values have been settled as 10% and 2.5% respectively (rounded to integer). The Pi value has been calculated for RIP and also for NRIP where the gene was present in a comparable number of species. These outputs have been plotted using Excel. All other graphics, where not explicitly stated, were generated with R 46 .

Conclusion
The results of this study highlight the presence of a defined sub-group of Rosaceae infecting species taxonomically and genetically related, with a high number of conserved core genes. The importance of the siderophores uptake genes has been extended to the complete transport system of ferrioxamine, with the identification of two genes present exclusively in the strains infecting the Rosaceae. These genes, namely fhuD and sidE, code for two proteins that require further studies and are possible new targets for development of novel control measures against RIP. Our results raise the interest towards E. tasmaniensis for a better understanding of the transition between non-pathogenicity and pathogenicity. Moreover, our data confirm the classification of Erwinia spp. Ejp617 as E. pyrifoliae.