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Article
Two catechol siderophores, acinetobactin and amonabactin,
are simultaneously produced by Aeromonas salmonicida
subsp salmonicida sharing part of the biosynthetic pathway
Miguel Balado, Alba Souto, Ana Vences, Valeria Careaga, Katherine Valderrama, Yuri
Segade, Jaime G. Rodrguez, Carlos R Osorio, Carlos Jimnez, and Manuel L. Lemos
ACS Chem. Biol., Just Accepted Manuscript DOI: 10.1021/acschembio.5b00624 Publication Date (Web): 13 Oct 2015
Downloaded from http://pubs.acs.org on October 16, 2015

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9 5 Two catechol siderophores, acinetobactin and amonabactin, are
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11 simultaneously produced by Aeromonas salmonicida subsp
12 salmonicida sharing part of the biosynthetic pathway
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10 Miguel Balado, Alba Souto, Ana Vences, Valeria P. Careaga, Katherine
17 Valderrama, Yuri Segade, Jaime Rodrguez, Carlos R. Osorio, Carlos Jimnez,
18 Manuel L. Lemos*
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20 Department of Microbiology and Parasitology, Institute of Aquaculture, Universidade de
21 Santiago de Compostela, Campus Sur, Santiago de Compostela 15782, Spain

22 Centro de Investigacins Cientficas Avanzadas (CICA), Departamento de Qumica
23 Fundamental, Facultade de Ciencias, Universidade da Corua, 15071 A Corua, Spain.
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*Corresponding author, Email: manuel.lemos@usc.es
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46 KEYWORDS: Aeromonas, Aeromonas salmonicida, iron uptake, siderophores,
47 acinetobactin, amonabactin
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3 ABSTRACT
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6 The iron uptake mechanisms based on siderophore synthesis used by the fish pathogen
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8 5 Aeromonas salmonicida subsp. salmonicida are still not completely understood and the
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precise structure of the siderophore(s) is unknown. The analysis of genome sequences
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11 revealed that this bacterium possesses two gene clusters putatively involved in the synthesis
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13 of siderophores. One cluster is a candidate to encode the synthesis of acinetobactin, the
14 siderophore of the human pathogen Acinetobacter baumannii, while the second cluster
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16 10 shows high similarity to the genes encoding amonabactin synthesis in Aeromonas
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18 hydrophila. Using a combination of genomic analysis, mutagenesis, biological assays,
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chemical purification and structural determination procedures, here we demonstrate that
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21 most A. salmonicida subsp salmonicida strains produce simultaneously the two siderophores,
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23 acinetobactin and amonabactin. Interestingly, the synthesis of both siderophores relies on a
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15 single copy of the genes encoding the synthesis of the catechol moiety (2,3
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26 dihydroxybenzoic acid) and on one encoding a phosphopantetheinyl transferase. These
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28 genes are present only in the amonabactin cluster, and a single mutation in any of them
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abolishes production of both siderophores. We could also demonstrate that some strains,
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31 isolated from fish reared in seawater, produce only acinetobactin since they present a
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33 20 deletion in the amonabactin biosynthesis gene amoG. Our study represents the first evidence
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of simultaneous production of acinetobactin and amonabactin by a bacterial pathogen, and
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36 reveals the plasticity of bacterial genomes and biosynthetic pathways. The fact that the same
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38 siderophore is produced by unrelated pathogens highlights the importance of these systems
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and their interchangeability between different bacteria.
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3 INTRODUCTION
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5 Iron is an essential element for the metabolism of most microorganisms, but due to its
6 physicochemical properties it is hard to find in assimilable forms in most biological
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8 environments. This is also true for animal tissues were most iron is tightly bound to iron
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10 5 containing proteins. Thus, bacteria have evolved a number of sophisticated mechanisms to
11 obtain iron from the environment and from their hosts in the case of pathogens.1 The
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13 mechanisms for acquisition of iron are generally recognized as essential factors in the
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15 survival of most bacterial pathogens within their hosts, and it is now clear that they
16 significantly contribute to bacterial virulence, being key factors for infectious diseases
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18 10 development.2, 3
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20 One of the main strategies of microorganims to acquire iron is the synthesis and
21 secretion of siderophores, lowmolecularweight structurally diverse Fe(III) chelators,
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23 which efficiently remove iron from ironbinding proteins or other ironcontaining
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25 compounds, and that then enter the cell through specific cell envelope transport proteins,
26 15 including outer membrane cognate receptors.2 Synthesis of siderophores is a complex
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28 process that usually requires a complete set of dedicated genes that encode the required
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30 biosynthetic enzymes. Many siderophores are usually synthesized by NRPS (nonribosomal
31 peptide synthetases), a multimodule and multicatalytic type of enzymes that assemble the
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33 different residues that form the final compound.4, 5, 6 From the amino acid sequence of these
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35 20 enzymes and through sequence comparisons with other known NRPSs it is usually feasible,
36 to some extent, to predict the residues that will be incorporated into the siderophore that they
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38 synthesize, being hence possible to obtain some important clues about the structure of the
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40 compound.7, 8, 9 All catecholtype siderophores contain DHBA (2,3dihydroxybenzoic acid),
41 which is formed from chorismic acid by a 4step biosynthetic pathway requiring the
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43 25 presence of 4 genes (entABCE in Escherichia coli) encoding the corresponding enzymes.
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45 These genes are usually well conserved among many different bacteria.4
46 Aeromonas salmonicida subsp salmonicida (hereafter A. salmonicida) is a Gram
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48 negative proteobacteria identified as the causative agent of furunculosis, a devastating
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50 disease affecting cultured and wild fish worldwide. The disease causes significant economic
51 30 losses in cultivated salmonids in fresh and marine waters and also affects a variety of non
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53 salmonid fish.10 Although siderophore production in this bacterium was known for more
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55 than 30 years,11, 12, 13
very little information is available about the chemistry of the
56 siderophore(s) produced and the genetics underlying its synthesis. 14
In a previous work we
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58 demonstrated that A. salmonicida produces at least one catechol siderophore related to
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3 anguibactin and/or acinetobactin, and provided evidences that the siderophore might contain
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5 a histamine moiety group.15 We demonstrate that this system was encoded on a
6 chromosomal gene cluster (asbGFDCBI) with high similarity to genes related to the
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8 synthesis of the siderophore acinetobactin, from the human pathogen Acinetobacter
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10 5 baumannii.16, 17 This gene cluster was present in many strains of A. salmonicida isolated
11 from different origins.15, 18
However, the completion of the genome sequence of A.
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13 salmonicida19 revealed the presence of a second cluster containing genes with high
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15 similarity to those involved in the synthesis and transport of amonabactin in A. hydrophila20,
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, and that also contains the genes necessary for DHBA biosynthesis. These two gene
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18 10 clusters would also encode two different outer membrane siderophore receptors (FstB and
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20 FstC, respectively) that were identified as ironregulated proteins in a proteomic study in A.
21 salmonicida.22
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23 In the present study, we report the identification of the siderophores produced by A.
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25 salmonicida which resulted to be acinetobactin and amonabactin, and demonstrate that each
26 15 of the two gene clusters encodes in fact the synthesis of the respective siderophore,
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28 confirming the former prediction based on the analysis of the genome of A. salmonicida.
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30 Furthermore, we also demonstrated that most strains tested produce both siderophores
31 simultaneously while certain strains produce only acinetobactin due to a mutation in one of
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33 the amonabactin biosynthetic genes.
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37 RESULTS AND DISCUSSION
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A search of the complete genome sequence of A. salmonicida subsp salmonicida strain
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A449 revealed the presence of two gene clusters that show homology to pseudomonine
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42 and acinetobactin (ORFs ASA_4368 to ASA_4380) (Table 1) and to amonabactin (ORFs
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25 ASA_1838 to ASA_1851) siderophore systems (Table 2). The two gene clusters seem to
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45 include the functions necessary for the synthesis of these siderophores and for the ferri
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47 siderophores transport through the corresponding outer membrane receptors. Both clusters
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are also present in all the A. salmonicida subsp. salmonicida strains whose genome
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50 sequences are deposited in the GenBank, and with a nucleotide sequence identity higher
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52 30 than 99% among strains 23.
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55 Analysis of the pseudomonine/acinetobactin cluster. The gene cluster spanning from
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57 ORF ASA_4368 to ASA_4380 contains the asbGFDCBI genes that we have previously
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3 described in strain RSP74.1 as genes involved in the synthesis of an uncharacterized
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5 catechol siderophore derived from histamine.15 This cluster also contains other genes
6 putatively involved in siderophore transport, including the outer membrane receptor FstB
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8 (Figure 1). According to in silico predictions using BLAST comparisons (Table 1) and the
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10 5 Pfam database,24 this cluster might encode the synthesis of a siderophore closely related to
11 pseudomonine, a phenolate siderophore produced by Pseudomonas entomophila.25 Indeed,
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13 the genes of this cluster show high similarity to pseudomonine genes and an identical gene
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15 arrangement (Figure 1, Table 1). However, the asb cluster lacks psmC, psmE and psmB
16 homologues, three genes that encode functions essential to synthesize activated salicylate
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18 10 from chorismate. In fact, a pmsCEAB mutant of P. entomophila showed a complete loss of
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20 pseudomonine production.25 In contrast, from our previous works we know that A.
21 salmonicida clearly produces DHBA,15 an essential component of all catechol siderophores,
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23 which indicates that the siderophore produced might have a catechol functional group. It is
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25 interesting to note that although the DHBA synthesis genes are usually linked to other genes
26 15 required for synthesis of catechol siderophores, the asb cluster of A. salmonicida does not
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28 contain genes encoding the synthesis of DHBA. All these observations clearly suggest that
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30 in A. salmonicida these genes must necessarily be located in another position in the
31 chromosome.
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33 The A. salmonicida asb cluster also shows high similarity (Figure 1, Table 1) to the
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35 20 cluster encoding the synthesis of acinetobactin,17, 26, 27 a catecholhydroxamate siderophore
36 that was first described as the siderophore of the human pathogen Acinetobacter
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38 baumannii,16 and so far not found in any other pathogen. Pseudomonine and acinetobactin
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40 have similar structures with the only change of a functional group, salicylate in
41 pseudomonine and DHBA in acinetobactin.28 A. baumannii acinetobactin cluster harbors the
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43 25 genes necessary for DHBA synthesis,26, 27
except the gene entA, that encodes the 2,3
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45 dihydro2,3dihydroxybenzoate dehydrogenase, which is located outside the cluster.29
46 When comparing the genomes of A. salmonicida and A. hydrophila (Figure 1), it is
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48 evident that the acinetobactin cluster represents a clear-cut insertion between fhuE gene
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50 (encoding a permease for ferrichrome uptake) and trmE gene (encoding a putative tRNA
51 30 modification GTPase), both common to all species of the genus Aeromonas. As mentioned
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53 above, the acinetobactin and pseudomonine gene clusters are synthenic and their encoded
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55 proteins show high amino acid similarity values (63 to 90%). However their G-C content
56 differs (58% for asb cluster vs 64% for pseudomonine cluster), showing the asb gene cluster
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58 the same G-C content than the rest of the A. salmonicida genome (ca. 58%). Altogether
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3 these results suggest that, although pseudomonine and acinetobacin genes would share a
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5 common origin, the acinetobactin gene cluster was likely acquired through an ancient gene
6 transfer event at some point during the evolution of A. salmonicida. The horizontal transfer
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8 of gene clusters encoding the synthesis and transport of siderophores seems to be a common
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10 5 feature in many bacteria.30, 31, 32, 33
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13 Analysis of the siderophore produced by A. salmonicida RSP74.1. In order to confirm the
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15 functionality of the asb cluster as well as the structure of the siderophore produced by A.
16 salmonicida strains harboring it, we isolated, purified and chemically characterized the
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18 10 siderophore present in cell free supernatants from a culture of strain RSP74.1 grown under
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20 iron deficient conditions. We know that this strain produces only one siderophore encoded
21 by the asb cluster since a mutation in asbD gene (Figure 1) completely abolishes
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23 siderophore production.15
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25 To accelerate the activity assayguided isolation of the siderophores, we employed
26 15 the methodology based on HLB (hydrophilic-lipophilic balance) cartridges and MS (mass
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28 spectrometry) that we successfully applied in the isolation of piscibactin9 and reisolation of
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30 vanchrobactin.34 Thus, SPE (solid phase extraction) of the cellfree culture supernatants of
31 strain RSP74.1 gave several fractions which were tested for siderophore activity and (+)
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33 LRESIMS. The fraction eluted with acetonitrile/water (8:2) each containing 0.1% TFA (v/v),
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35 20 resulted to be siderophore active and showed the presence of an intense peak at m/z 347 ([M
36 + H]+), suggesting the presence of acinetobactin (1) (Figure 2) as it was predicted by the
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38 genomic data described above. Final purification of this fraction by HPLC allowed us to
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40 isolate a pure compound which UV and NMR spectral data (Figure 3), HRESIMS and
41 optical rotation matched with those reported for acinetobactin in 199416 (Supplementary
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43 25 Figures S1S5). In 2009, it was suggested that the reported structure was an unstable
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45 intermediate, named as preacinetobactin, and the correct structure was proposed as 1.28 The
46 structure of acinetobactin as 1, including its absolute configuration, was confirmed by
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48 chemical synthesis.35 In addition, we found that the pure compound isolated could be used
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50 as a siderophore by A. salmonicida strain RSP74.1asbD in plate bioassays (data not
51 30 shown). Furthermore, acinetobactin was not detected in the supernatants of RSP74.1asbD
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53 mutant. Thus, all these results clearly demonstrate that A. salmonicida produces at least
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55 acinetobactin as siderophore when growing in ironlimiting conditions. To the best of our
56 knowledge this is the first report of acinetobactin acting as siderophore in bacteria outside
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58 the Acinetobacter genus.
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5 Analysis of the gene cluster involved in amonabactin biosynthesis. The analysis of the A.
6 salmonicida A449 genome revealed a second cluster (ORFs ASA_1838 to ASA_1851) that
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8 contains genes with high similarity to those involved in the synthesis and transport of
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10 5 amonabactin, a catechol isolated from A. hydrophila,20, 21 and that would include the genes
11 necessary for DHBA biosynthesis (entCEBA).36 It also includes the genes necessary for
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13 siderophore transport (Figure 4). Closed homologues of these genes are extensively present
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15 in all Aeromonas species for which genomic data are available. We renamed the genes
16 putatively involved in siderophore assembly as amoF, amoG, amoH (encoding non
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18 10 ribosomal peptide synthetases) and amoD, that would encode a phosphopantetheinyl
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20 transferase. The percentage of similarity of the A. salmonicida proteins with the A.
21 hydrophila homologues are above 90% in most of the cases (Table 2). The only noticeable
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23 difference between the two species is that in A. hydrophila the genes encoding the transport
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25 proteins, including the outer membrane receptor, are located in a different locus in the
26 15 chromosome (Figure 4).
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28 Since, as shown above, strain RSP74.1 produces only acinetobactin, we selected
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30 another highly virulent strain of A. salmonicida (VT45.1) to study the amonabactin cluster
31 functions. Using the allelic exchange procedure we constructed several mutants in strain
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33 VT45.1 and test the resulting phenotype for growth in iron deficient conditions, as well as
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35 20 DHBA and siderophore production. We first mutated the asbD gene of the acinetobactin
36 cluster in this strain, abolishing acinetobactin synthesis. This mutant showed a phenotype
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38 almost identical to the wildtype strain, as it was capable of growth under ironlimiting
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40 conditions, produced about the same amount of catechols and displayed almost the same
41 siderophore activity in the CAS test (Figure 5). This suggests that another siderophore is
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43 25 being produced. When we constructed a single deletion mutant of the amoG gene of the
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45 amonabactin cluster, we again found that the mutant strain behaved similarly to the parental
46 strain (Figure 5), indicating that acinetobactin was synthesized. However, when we deleted
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48 both genes (asbD and amoG), we observed that the double mutant was almost unable to
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50 grow in ironlimiting conditions and the siderophore activity was very low (Figure 5).
51 30 Overall these results suggest that strain VT45.1 produces simultaneously acinetobactin and
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53 likely amonabactin. In addition, our results clearly suggest that strain RSP74.1, despite
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55 harboring an amonabactin synthesis cluster, must likely carry an inactivating mutation in
56 one or more of the genes involved in amonabactin biosynthesis.
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3 Identification of amonabactins in A. salmonicida VT45.1. From the results described
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5 above, there are evidences that VT45.1 produces another siderophore in addition to
6 acinetobactin, and according to in silico predictions this siderophore might be amonabactin.
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8 Therefore, in order to demonstrate that amonabactin is being made by A. salmonicida, we
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10 5 used the mutant strain VT45.1asbD (impaired in the synthesis of acinetobactin) to isolate
11 the siderophore produced under ironlimiting growth conditions. A cellfree culture
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13 supernatant of this strain was subjected to a similar procedure described above for
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15 acinetobactin isolation. This method allowed us to isolate two pure compounds which were
16 identified as amonabactin P750 (2) and amonabactin P693 (3) (Figure 2) by comparison of
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18 10 their NMR (Figure 3) and HRESIMS data with those reported to these compounds.37
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20 Furthermore, incubation of the cellfree culture supernatants of VT45.1asbD strain with
21 FeCl3*6H2O and GaBr3, posterior fractionation as described above and HPLCHRESIMS in
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23 the negative mode experiments, allowed us to detect the Fe(III) complexes for amonabactin
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25 P750 (2), P693 (3), T789 (4) and T732 (5) and Ga(III) complexes for amonabactin P750 (2)
26 15 and P693 (3) (Suplementary Figures S6S14; Table S1). The pure compounds isolated could
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28 be used as siderophores by A. salmonicida strain VT45.1asbD as demonstrated by agar
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30 plate bioassays (data not shown). Thus, we could demonstrate that A. salmonicida produces
31 also amonabactins as siderophores when growing in ironlimiting conditions.
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33 Furthermore, to demonstrate that both siderophores are co-produced at the same time,
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35 20 we used supernatants from wild type strain VT45.1 to detect the simultaneous presence of
36 acinetobactin and amonabactins. The results of LC-MS experiments show that both
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38 siderophores are present in the extracellular medium of cells grown under iron limitation
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40 (Suplementary Figures S15 and S16). Thus, A. salmonicida VT45.1 and similar strains
41 clearly produce, under laboratory conditions, acinetobactin and amonabactins
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43 25 simultaneously.
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46 Acinetobactin and amonabactin synthesis share biosynthetic enzymes. The synthesis of
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48 catechol siderophores begins with the conversion of chorismate in DHBA. Subsequently, the
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50 NRPS selects the different siderophore precursors to form the final structure.4 In previous
51 30 sections we have shown that asbD and amoG encode essential NRPSs that participate
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53 respectively in acinetobactin and amonabactin biosynthesis. To convert chorismate into
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55 DHBA entABC homologues are necessary. Once DHBA is synthesized, an EntE homologue
56 begins the assembly process.4 In the genome of A. salmonicida 449 we found homologues of
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58 all these genes only within the amonabactin cluster (Figure 4; Table 2), being absent in the
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3 acinetobactin cluster, which suggests that acinetobactin and amonabactin production share
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5 the DHBA synthesis pathway. To test this hypothesis, DHBA and siderophore production
6 were analyzed in a entB mutant from strain VT45.1.
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8 Interestingly, when we deleted entB from the amonabactin cluster, the mutant was
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10 5 unable to produce any siderophore. In addition, analysis of the supernatants by the Arnow
11 test revealed that DHBA production was completely abolished (Figure 5). Complementation
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13 of the mutation, by providing in trans an intact copy of entB gene, restored both DHBA and
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15 siderophore production (Figure 5). This demonstrates that synthesis of acinetobactin and
16 amonabactin depends on the same set of genes necessary for DHBA synthesis, and that there
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18 10 are not other genes that can be used instead. In fact, an in silico search on the genome of
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20 strain A449 does not show any putative orthologs for entCEB or entA genes.
21 In addition, an in silico analysis of the catalytic domains present in the asb cluster
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23 shows the lack of a phosphopantetheinyl transferase (EntD homologue), an essential
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25 component for activation of PCP domains of the NRPS.4 In the genome of A. salmonicida
26 15 there is a single copy of an entD homologue, and this gene is located within the amonabactin
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28 gene cluster (ASA1846, renamed to amoD). Interestingly, when we mutated this gene in
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30 strain VT45.1, the resulting phenotype was almost identical to the entB mutant (Figure 5).
31 Albeit the amoD mutant produces levels of DHBA similar to the parental strain, it was
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33 unable to grow under iron limitation and the amount of siderophores produced was
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35 20 equivalent to that of the double mutant amoGasbD. Complementation of the mutation, by
36 providing in trans a copy of amoD gene, fully restored the wild type phenotype (Figure 5).
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38 All this suggest that amoD is an essential gene for acinetobactin and amonabactin synthesis
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40 and that it is the only gene encoding this function in A. salmonicida. It is noteworthy that the
41 entD homologue is also absent from the gene cluster encoding the synthesis of
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43 25 pseudomonine in Pseudomonas entomophila, being located in a region of the genome
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45 unrelated to siderophore synthesis.25
46 Based on the results reported above, and on in silico predictions upon analysis of the
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48 protein sequences and domains, a proposed biosynthetic pathway for both siderophores in A.
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50 salmonicida is depicted in Figure 6. Once DHBA is made from chorismate using EntCBA
51 30 enzymes, part of it would be used by AsbBCD proteins to synthesize acinetobactin from
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53 histidine and threonine. At the same time DHBA could be used by enzymes AmoFGH for
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55 the synthesis of amonabactins from lysine and phenylalanine (amonabactins P) or
56 tryptophan (amonabactins T)37 (Figure 2).
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3 Some strains harbor an inactivated copy of amoG gene. We have previously shown that
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5 singlegene mutations within asb cluster in strain RSP74.1 abolish siderophore
6 production,15 suggesting that this strain produces only one type of siderophore encoded by
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8 this cluster. In the present work we demonstrated that strain RSP74.1 produces acinetobactin
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10 5 as the only siderophore and, although harboring the amonabactin cluster, it is unable to
11 produce amonabactin.
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13 In order to find the reason for this impairment, we fully sequenced the
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15 RSP74.1 (accession number KM262645) and VT45.1 (accession number KM262646)
16 amonabactin gene clusters, and compared them with the homologous sequence in the
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18 10 genome of strain A449 deposited in GenBank. The results clearly showed that RSP74.1
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20 amoG gene (that encodes the main NRPS involved in amonabactin biosynthesis) has a
21 deletion of 43 bp at the 3 end of the gene (Supplementary Figure S17) that would lead to a
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23 truncated AmoG protein. An in silico analysis of the resulting catalytic domains shows that
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25 this truncated protein would not have the PCP domain at the Cterminal end (Figure 7),
26 15 making it unable to complete the synthesis of amonabactin. This domain is thus intact in
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28 strains A449 and VT45.1 and, as shown above, strain VT45.1 clearly produces both
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30 amonabactin and acinetobactin.
31 To assess whether the presence of this deletion in amoG gene is a unique feature of
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33 strain RSP74.1 or if it also occurs in other strains, we tested by PCR the presence of this 43
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35 20 bp deletion in a collection of 20 A. salmonicida strains isolated from a variety of diseased
36 fish. As a result, we found that this feature is present in several strains of this pathogen
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38 (Table 3). Interestingly, we could evidence the existence of a correlation between the origin
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40 of the strains and the presence of the 43 bp deletion, since all strains that lack this small
41 fragment were isolated from marine fish or fish reared in seawater. Conversely, all the
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43 25 strains producing the two siderophores were isolated from freshwaterreared fish. This
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45 deletion was not detected in any of the genomes of A. salmonicida subsp. salmonicida
46 deposited in the GenBank, suggesting that strains harboring the amoG deletion might have a
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48 clonal origin. The biological or ecological significance of this deletion that impairs
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50 amonabactin synthesis, and its possible restriction to the marine environments, would need
51 30 further studies in order to find a plausible explanation.
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54
55 CONCLUSIONS
56 From the results reported here and from the genomic data available at GenBank, we can
57
58 conclude that the amonabactin synthesis gene cluster is widespread among all species of the
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3 genus Aeromonas, while acinetobactin synthesis is restricted to A. salmonicida, being
4
5 present in all strains and subspecies for which genomic data are available: salmonicida,
6 achromogenes, pectinolytica and masoucida. From the analysis of the genomic context of
7
8 acinetobactin cluster, seems clear that this gene cluster was likely acquired through
9
10 5 horizontal gene transfer at some point during the speciation process of A. salmonicida, as
11 demonstrated by its insertion between two genes, fhuE and trmE, that are common to all
12
13 species of the genus Aeromonas. It is noteworthy that the acinetobactin cluster in A.
14
15 salmonicida does not harbor the DHBA synthesis genes, being the synthesis of acinetobactin
16 dependent of the DHBA synthesis enzymes, as well as of the Phosphopantetheinyl
17
18 10 Transferase encoded by the amonabactin cluster. This feature reinforces the idea that
19
20 amonabactin is the ancestral siderophore of the species, and that acinetobactin was later
21 acquired from other bacteria. The evolutionary advantage of having two siderophore
22
23 systems is unclear. Although the strains of A. salmonicida having both clusters functional
24
25 are highly virulent, the strains deficient in amonabactin synthesis were also isolated as
26 15 causative agents of furunculosis. It has been suggested that the apparent redundancy of iron
27
28 uptake mechanisms could be an advantage in changeable environments.38, 39 We could also
29
30 speculate that each one of the siderophores will be preferentially used in specific
31 environments with different iron availability, such as water (freshwater or seawater) and the
32
33 host tissues. Experiments currently under way will help to determine whether both systems
34
35 20 are actually expressed during infection or whether one of them is switched off at some stage
36 inside the host. Future work will also elucidate the precise role of each one of these iron
37
38 uptake systems in the biology and the virulence of A. salmonicida subsp salmonicida.
39
40
41
42 METHODS
43
25 Bacterial strains, plasmids and media. Strains and plasmids used, as well as those derived
44
45 from this study are listed in Supplementary Table S2. A. salmonicida subsp. salmonicida
46
47 strains were grown at 22 C in Tryptic Soy Agar and Broth (Pronadisa) supplemented with
48
1% NaCl (TSA1 and TSB1, respectively), as well as in M9 minimal medium40
49
50 supplemented with 0.2% Casamino Acids (Difco) (CM9). Escherichia coli strains were
51
52 30 routinely grown at 37 C in Luria Bertani (LB) medium (Pronadisa) or LB supplemented
53
54
with the appropriate antibiotics. Ampicillin sodium salt (Ap) (SigmaAldrich) was used at
55 50 g mL-1; tetracycline (Tc), at 12 g mL-1 and gentamicin (Gm) at 15 g mL-1 (final
56
57 concentrations). All stocks were filter sterilized and stored at 20C. The iron chelator 2,2
58
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3 dipyridyl (TCI) was dissolved in distilled water to prepare a stock solution at 20 mM that
4
5 was added to the sterile media at appropriated concentrations.
6
7 DNA manipulations and Bioinformatics tools. Total genomic DNA from A. salmonicida
8
9 was purified with the EasyDNA kit (Invitrogen). Plasmid DNA purification and extraction
10 5 of DNA from agarose gels were carried out using kits from Fermentas (ThermoFisher).
11
12 PCR reactions were routinely carried out in a TGradient Thermal Cycler (Biometra), with
13
14 Taq polymerase BioTaq (Bioline). DNA sequences were determined by the dideoxy chain
15 termination method using the CEQ DTCSQuick Start Kit (Beckman Coulter) using a
16
17 capillary DNA sequencer CEQ 8000 (Beckman Coulter). The NCBI services
18
19 10 (http://ncbi.nlm.nih.gov) were used to consult the DNA and protein sequence databases with
20 BLAST algorithm 41
. Prediction of protein domains was carried out by using the Pfam
21
22 database online facilities (http://www.sanger.ac.uk/Software/Pfam/).24
23
24
25
Construction of mutants by allelic exchange. Inframe deletion of asbD, amoG, entD
26 (amoD) and entB genes in A. salmonicida subsp. salmonicida VT45.1 were constructed as
27
28 15 previously described.15 PCR amplifications of two fragments of each gene and flanking
29
30
regions (primers listed in Supplementary Table S3), when ligated together result in an in
31 frame (nonpolar) deletion.. Each deleted allele construction was ligated into the suicide
32
33 vector pKEK229 42, that requires the pir gene product for replication, and contains the sacB
34
35
gene that confers sucrose sensivity. The resulting plasmids were mated from E. coli S17-1-
43
36 20 pir into A. salmonicida subsp. salmonicida VT45.1 wild type strain (TcR) and into
37
38 previously constructed mutant strains, and exconjugants with the plasmid (ApR) integrated
39
40 in the chromosome by homologous recombination were selected. A second recombination
41 event was obtained by selecting for sucrose (15%) resistance and further checking for
42
43 plasmid loss and for allelic exchange. This process led to the generation of A. salmonicida
44
45 25 subsp. salmonicida MB158 (amoG), MON120 (asbD), MB168 (amoD) and AVL1
46 (entB) individual mutant strains and the double mutant MB160 (asbDamoG)
47
48 (Supplementary Table S2). DNA sequencing of the region involved in the deletion was
49
50 carried out to ensure that all mutations were inframe. For entB and amoD gene
51 complementation the ORFs were PCR-amplified (primers listed in Supplementary Table S3)
52
44
53 30 with Hi-Fidelity Kapa Taq (Kapa), cloned into pHRP309 vector and mobilized from E.
54
55 coli S17-1 -pir into the respective A. salmonicida defective mutant. Gene amoD was
56 cloned along with its own promoter (strain MB272), while entB was cloned under control of
57
58 the Vibrio anguillarum fvtA promoter 45 (strain MB273).
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3 Growth under iron limiting conditions and siderophore production. To test the ability
4
5 of A. salmonicida subsp. salmonicida mutants to grow under iron limited conditions,
6 overnight cultures in LB of the parental and mutant strains were adjusted to an OD600=0.5 in
7
8 a UV/VIS spectrophotometer (Hitachi) and diluted 1:100 in CM9 medium containing 75 M
9
10 5 2,2dipyridyl. Cultures were incubated at 25 C with shaking at 150 rpm and growth
11 (OD600) was recorded after 18 h incubation. Siderophore production was measured using the
12
46
13 chrome azurolS (CAS) liquid assay . Equal volumes of culture supernatants and CAS
14
15 solution were mixed and the A630 was measured in the spectrophotometer. In addition, the
16 Arnow test 47
was used for spectrophotometric determination of DHBA concentration. A
17
18 10 noninoculated CM9 sample containing 2,2dipyridyl at appropriate concentration was
19
20 used as spectrophotometric blank and as negative control for CAS and Arnow assays.
21 Siderophore production for purification and structural analysis (see below) was achieved by
22
23 growing appropriate strains in 1 L of CM9 plus 50 M 2,2dipyridyl in 2 L flasks with
24
25 continous shaking at 25 C for 24 h. After incubation, bacterial cells were pelleted by
26
15 centrifugation at 10,000 rpm in a Beckman J21 high speed centrifuge. Supernatants were
27
28 filtered through a continuous filtration cartridge with a 0.45 m pore size membrane
29
30 (Millipore) to remove any cell and organic debris. Siderophore activity present in
31
32 supernatants was evaluated using the CAS assay.
33
34 Extraction, purification and characterization of siderophores
35
36 20 Isolation of acinetobactin (1). One litre of cellfree culture broth of Aeromonas
37
38 salmonicida subsp. salmonicida strain RSP74.1 was lyophilized to give 17 g. One gram of
39 this material was dissolved in water (1 mL) and loaded in a OASIS HLB cartridge
40
41 (Waters) (35 cm3, 6 g), which was previously conditioned and equilibrated with 60 mL of
42
43 acetonitrile (solvent B) and 60 mL of water (solvent A), each containing 0.1% TFA (v/v). It
44 25 was eluted with the following mixtures of solvent A and solvent B: 1:0, 9:1, 8:2, 7:3 and
45
46 0:1.After washing the cartridge with 60 mL of solvent A and 30 mL of mixture of solvents
47
48 A:B (9:1), the fraction eluted with 60 mL of a mixture of solvents A:B (8:2) (6.4 mg after
49 evaporation to dryness) showed the presence of acinetobactin (1) by the (+)ESIMS
50
51 displaying an intense peak at m/z 347 ([M + H]+). Final purification of 2 mg of that fraction
52
53 30 by HPLC using a Discovery HS F5 (100 x 4.6 mm, 5 m) column with a mobile phase
54
consisting of a 40 min gradient from 10 to 50% of solvent B at a flow rate of 1 mL min-1
55
56 gave 0.8 mg of pure acinetobactin (1) isolated as its TFA salt. From 4 g of the initial
57
58 lyophilized material, repeating the same procedure, 13.4 mg of pure acinetobactin (1) were
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3 isolated. Compound confirmation was carried out by UV, 1H NMR and HRESIMS.
4
5 Acinetobactin (1): []D23= - 29.7 (c = 0.65, MeOH,); 1H and 13C NMR (Supplementary
6
Table S4). (+)-LR-ESIMS m/z: 347.14 [M + H]+. (+)-HR-ESIMS m/z: 347.1362 [M + H]+
7
8 (calcd. for C16H19N4O5, 347.1350).
9
10
11 5 Isolation of amonabactins P750 (2) and P693 (3). One litre of a cellfree culture broth of
12 A. salmonicida strain VT45.1asbD was lyophilized to give 13.2 g. Part of this material
13
14 (5.77 g) was dissolved in 6 mL of water, divided in three batches (2 mL each) and loaded
15
16 into three OASIS HLB cartridges (35 cm3, 6 g), which were previously conditioned and
17
equilibrated with 60 mL of acetonitrile (solvent B) and 60 mL of H2O (solvent A), each
18
19 10 containing 0.1% TFA (v/v). After washing each cartridge with 60 mL of solvent A, several
20
21 fractions were eluted with a gradient mixture of solvents A and B: (9:1), (8:2), (7:3), (1:1)
22
and (0:1). Fraction eluted with 60 mL of a mixture of solvents A:B (1:1) gave 54.3 mg of
23
24 residue after lyophilization. Final purification of this fraction was achieved by HPLC using a
25
26 Discovery HS F5 (100 x 4.6 mm, 5 m) column at a flow rate of 1 mL min-1 with a mobile
27
28
15 phase consisting of a mixture of water (solvent A) and acetonitrile (solvent B), each
29 containing 0.1% TFA (v/v): 15 min gradient from 10 to 60% of solvent B, 5 min gradient
30
31 from 60 to 100% of solvent B, and finally 2 min isocratic period at 100% of solvent B.
32
33
Fractions containing the first siderophore were pooled and dried in vacuo to afford 5.8 mg of
34 amonabactin P750 (2). Other fractions containing the second siderophore were pooled and
35
36 20 then purified by HPLC using a Discovery HS F5 (100 x 4.6 mm, 5 m) column at a flow
37
38 rate of 1 mL min-1 with a mobile phase consisting of an isocratic mixture of 35% of
39 acetonitrile in water (v/v, each solvent containing 0.1% TFA) to give 0.5 mg of amonabactin
40
41 P693 (3). The structures of both compounds were confirmed by UV, NMR and HRESIMS
42
43 which spectral data are in concordance with those published for these compounds.37
44 25 Amonabactin P750 (2): []D20= + 5.8 (c =0.14, MeOH); (+)HRESIMS: m/z 751.3286 [M +
45
46 H]+ (calcd. for C37H47N6O11, 751.3297). Amonabactin P693 (3): []D20= + 59 (c =0.02,
47
48 MeOH); (+)HRESIMS: m/z 694.3048 [M + H]+ (calcd. for C35H44N5O10, 694.3083).
49
50 Analytical detection of amonabactins P750 (2), P693 (3), T789 (4) and T732 (5) and
51
52 their FeIII Complexes. One litre of cellfree culture broth from strain VT45.1asbD was
53
54 30 concentrated in vacuo to 0.5 L. A portion of 150 mL was transferred to a roundbottom
55 flask, 100 mg FeCl3*6H2O was added and the resulting mixture was gently stirred for 5 min.
56
57 After incubation at 4 C overnight, the solution was passed through two OASIS HLB
58
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3 cartridges (35 cm3, 6 g), which were previously conditioned and equilibrated with 60 mL of
4
5 acetonitrile (solvent B) and 60 mL of H2O (solvent A) and then, eluted with 30 mL using the
6 following mixtures of solvent A:B (9:1), (8:2), (7:3), (1:1), (3:7) and (0:1). All fractions
7
8 were concentrated in vacuo to 2 mL and submitted to Arnow and siderophore assays.
9
10 5 Fractions eluted with the mixture of solvents A:B (9:1) and (8:2) gave positive to those
11 assays. Then, they were analyzed by HPLCHRESIMS in the negative mode using a
12
13 Atlantis dC18 (100 x 4.6 mm, 5 m) column (Waters) at a flow rate of 1 mL min-1 with a
14
15 mobile phase consisting of a mixture of water and acetonitrile: 35 min gradient from 10 to
16 100% of acetonitrile, 2 min isocratic period at 100% of acetonitrile, and finally, 5 min from
17
18 10 100 to 10% of acetonitrile. Extracted mass experiments showed the presence of the
19
20 corresponding [Fe(Amonabactin)]- and [Fe(Amonabactin)]2- complexes for amonabactin
21 P750 (2), amonabactin P693 (3), amonabactin T789 (4) and T732 (5) (Supplementary
22
23 Figures S13, S14 and Table S1).
24
25
26
Analytical detection of amonabactins P693 and P750 as their GaIII Complexes. One
27 15 litre of cellfree culture broth from strain VT45.1asbD was concentrated in vacuo to 300
28
29 mL, adding 61 mg of GaBr3 and then, incubated at 4 C overnight. The resulting solution
30
31
was passed through five OASIS HLB cartridges (35 cm3, 6 g) using a similar procedure as
32 described before. Analysis by HPLCHRESIMS in the negative mode of the fractions eluted
33
34 from the HLB cartridges with a mixture of solvents A:B (9:1) and (8:2) showed the presence
35
36
20 of the corresponding [Ga(Amonabactin)]- and [Ga(Amonabactin)]2- complexes for
37 amonabactin P750 (2) and amonabactin P693 (3) (Supplementary Table S1).
38
39
40 Detection of acinetobactin and amonabactins in VT45.1 wild type strain. One litre of a
41 cell-free culture broth of VT45.1 wild-type strain was lyophilized to give 14,8 g of material.
42
43 One gram of this material was submitted to the same SPE methodology described above and
44
45 25 the resulting fractions were analyzed by HPLC-HRESIMS in the positive mode. Fraction
46 eluted with a mixture of solvents A:B (8:2) (29 mg after evaporation to dryness) showed the
47
48 presence of acinetobactin (1) (Suplementary Figure S15). Fraction eluted with a mixture of
49
50 solvents A:B (1:1) (5.5 mg after evaporation to dryness) showed the presence of
51 amonabactin P750 (2) and amonabactin P693 (3) (Suplementary Figure S16).
52
53
54 30 Monitoring of active fractions. Siderophore activity in the different fractions was followed
55
by the CAS test (see above) and by biological assays. For the CAS assay, equal volumes of
56
57 1:10 diluted fractions and CAS solution were mixtured and the A63046 was measured.
58
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9, 48
3 Biological activity was detected as previously described by testing the ability of
4
5 fractions or purified compounds to promote the growth of A. salmonicida indicator strains
6 innoculated in CM9 agar with 130 M 2,2dipyridyl (inhibitory concentration). All
7
8 fractions and compounds to be tested were adequately dissolved in aqueous solutions at a
9
10 5 concentration of 1 mg mL-1, and 10 L were used to impregnate blank paper disks (6 mm )
11
that were deposited in the CM9 agar plates. As indicator strain we used strain MON15
12
13 (RSP74.1asbD)15 for detection of acinetobactin and amonabactin since this strain does not
14
15 produce any of these siderophores, but can use both of them since it has intact the two outer
16
membrane receptors (FstB and FstC). A growth halo of the indicator strain around a
17
18 10 particular sample indicates siderophore activity.
19
20
21 ASSOCIATED CONTENT
22
23
Supporting Information
24
25
26 The Supporting Information is available free of charge on the ACS Publications website at
27 DOI:
28
29 Figures S1 to S5, chemical characterization of acinetobactin
30 Figures S6 to S14, chemical characterization of amonabactins
31 Figures S15 and S16, detection of simultaneous production of acinetobactin and
32 amonabactins in wild type strain VT45.1.
33
Figure S17, Partial DNA sequence of gene amoG showing a deletion of 43 bp present in
34
35 some strains.
36 15 Table S1. Identification of the amonabactin Ga(III) and Fe(III) complexes.
37 Table S2. Strains and plasmids used.
38 Table S3. Primers used for mutant construction, complementation and strain screening.
39 Table S4. NMR spectral data for acinetobactin-TFA salt.
40
41 Accesion codes
42
43 GenBank accesion codes for amonabactin cluster are KM262645 for strain RSP74.1 and
44
45 KM262646 for strain VT45.1.
46
47
48 AUTHOR INFORMATION
49 Corresponding Author
50
51 20 *Manuel L. Lemos, E-mail: manuel.lemos@usc.es. Phone: +34-881816080
52
53
54 ACKNOWLEDGMENTS
55
56 This work was supported by grant 10PXIB235157PR from Xunta de Galicia, Spain; and by
57
58
grants CSD2007-00002 (CONSOLIDER Aquagenomics) and AGL2012-39274-C02-01/02
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3 from Spanish Ministry of Economy and Competitiveness (MINECO), Spain, both cofunded
4
5 by the FEDER Programme from the European Union.
6
7
8 Notes
9
10 5 The authors declare no competing financial interest.
11
12
13
14
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12 vanchrobactinmediated iron uptake in Vibrio anguillarum: Evolution and ecology of
13 10 a fish pathogen. Environ. Microbiol. Rep. 2, 1926.
14 33. Osorio, C. R., Rivas, A. J., Balado, M., Fuentes-Monteverde, J. C., Rodriguez, J.,
15 Jimenez, C., Lemos, M. L., and Waldor, M. K. (2015) A transmissible plasmidborne
16 pathogenicity island encodes piscibactin biosynthesis in the fish pathogen
17 Photobacterium damselae subsp. piscicida. Appl. Environ. Microbiol. 82, In press.
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15 34. Espada, A., Anta, C., Bragado, A., Rodriguez, J., and Jimenez, C. (2011) An approach to
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20 speed up the isolation of hydrophilic metabolites from natural sources at
21 semipreparative level by using a hydrophilic-lipophilic balance/mixed-mode strong
22 cation exchange-high-performance liquid chromatography/mass spectrometry system.
23 J. Chromatogr. A. 1218, 17901794.
24 20 35. Takeuchi, Y., Ozaki, S., Satoh, M., Mimura, K., Hara, S., Abe, H., Nishioka, H., and
25 Harayama, T. (2010) Synthesis of acinetobactin. Chem. Pharm. Bull. (Tokyo). 58,
26 15521553.
27 36. Massad, G., Arceneaux, J. E., and Byers, B. R. (1994) Diversity of siderophore genes
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encoding biosynthesis of 2,3-dihydroxybenzoic acid in Aeromonas spp. Biometals. 7,
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30 25 227236.
31 37. Telford, J. R., Leary, J. A., Tunstad, L. M. G., Byers, B. R., and Raymond, K. N. (1994)
32 Amonabactin: Characterization of a series of siderophores from Aeromonas
33 hydrophila. J. Am. Chem. Soc. 116, 44994500.
34 38. Cordero, O. X., Ventouras, L. A., DeLong, E. F., and Polz, M. F. (2012) Public good
35 30 dynamics drive evolution of iron acquisition strategies in natural bacterioplankton
36 populations. Proc. Natl. Acad. Sci. U. S. A. 109, 2005920064.
37 39. Dumas, Z., Ross-Gillespie, A., and Kummerli, R. (2013) Switching between apparently
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redundant iron-uptake mechanisms benefits bacteria in changeable environments. Proc.
39
40 Biol. Sci. 280, 20131055.
41 35 40. Miller, J. H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory,
42 Cold Spring Harbor, N.Y.
43 41. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) Basic local
44 alignment search tool. J. Mol. Biol. 215, 403-410.
45 42. Correa, N. E., Lauriano, C. M., McGee, R., and Klose, K. E. (2000) Phosphorylation of
46 40 the flagellar regulatory protein FlrC is necessary for Vibrio cholerae motility and
47 enhanced colonization. Mol. Microbiol. 35, 743755.
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43. Herrero, M., de Lorenzo, V., and Timmis, K. N. (1990) Transposon vectors containing
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50 non-antibiotic resistance selection markers for cloning and stable chromosomal
51 insertion of foreign genes in gram-negative bacteria. J. Bacteriol. 172, 65576567.
52 45 44. Parales, R. E., and Harwood, C. S. (1993) Construction and use of a new broad-host-
53 range lacZ transcriptional fusion vector, pHRP309, for gram- bacteria. Gene 133, 23
54 30.
55 45. Balado, M., Osorio, C. R., and Lemos, M. L. (2009) FvtA is the receptor for the
56 siderophore vanchrobactin in Vibrio anguillarum: utility as a route of entry for
57 50 vanchrobactin analogues. Appl. Environ. Microbiol. 75, 27752783.
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3 46. Schwyn, B., and Neilands, J. B. (1987) Universal chemical assay for the detection and
4 determination of siderophores. Anal. Biochem. 160, 4756.
5 47. Arnow, L. E. (1937) Colorimetric determination of the components of 3,4-
6 dihydroxyphenyl-alanine-tyrosine mixtures. J. Biol. Chem. 118, 531537.
7 5 48. Soengas, R. G., Anta, C., Espada, A., Paz, V., Ares, I. R., Balado, M., Rodrguez, J.,
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Lemos, M. L., and Jimnez, C. (2006) Structural characterization of vanchrobactin, a
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10 new catechol siderophore produced by the fish pathogen Vibrio anguillarum serotype
11 O2. Tetrahedron Lett. 47, 71137116.
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6 Table 1. Description of the proteins encoded by the genes of the acinetobactin cluster (loci numbers correspond to the genome sequence of
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strain A449) in A. salmonicida, and their closest homologues.
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10 Aeromonas salmonicida subsp. salmonicida A449 chromosome a a a
A. hydrophila ATCC7966 Pseudomonas entomophila Acinetobacter baumannii
11 Locus (No. aa) Encoded Protein, description Pseudomonine system Acinetobactin system
12
ASA_4363 (701) FhuA, TonB-dependent ferrichrome receptor YP_858692.1 (93, 95)
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ASA_4364 (548) FhuB, ATP-binding component YP_858693.1 (94, 97)
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ASA_4365 (254) FhuC, ferrichrome ABC transporter ATP-binding protein YP_858694.1 (91, 94)
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ASA_4366 (308) FhuD, ferrichrome-binding periplasmic protein YP_858695.1 (80, 86)
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17 ASA_4367 (660) FhuE, ABC transporter permease subunit YP_858696.1 (88, 92)
18 ASA_4368 (824) FstA/B, TonB-dependent siderophore receptor YP_608099.1 (46, 63) ENW75959.1 (44, 61)
19 ASA_4369 (322) AsuB, ABC transporter, periplasmic protein YP_608100.1 (72, 82) ENW75958.1 (54, 71)
20 ASA_4370 (260) AsuE, ABC transporter, ATP-binding protein YP_608101.1 (74, 85) ENW75957.1 (69, 82)
21 ASA_4371 (316) AsuC, ABC transporter, permease YP_608102.1 (72, 86) ENW75956.1 (61, 78)
22 ASA_4372 (307) AsuD, ABC transporter, permease YP_608103.1 (79, 90) ENW75955.1 (72, 87)
23 ASA_4373 (589) AsuE, ABC transporter permease/ATP-binding protein YP_608104.1 (66, 76) ENW75966.1 (31, 49)
24 ASA_4374 (607) AsuF, ABC transporter permease/ATP-binding protein YP_608105.1 (68, 79) ENW75965.1 (31, 49)
25 ASA_4375 (165) AsbI, RNA polymerase sigma-70 factor YP_608106.1 (63, 77)
26 ASA_4376 (709) AsbB, non-ribosomal peptide synthetase YP_608107.1 (50, 67) ENW75953.1 (50, 66)
27 ASA_4377 (440) AsbC, L-lysine 6-monooxygenase YP_608108.1 (71, 82) ENW75960.1 (70, 82)
28 ASA_4378 (1453) AsbA/D, non-ribosomal peptide synthetase YP_608109.1 (61, 71) ENW75952.1 (41, 61)
29 ENW75961.1 (55, 69)
30 b
ASA_4379 (83) AsbF, 2,3-dihydroxybenzoate-AMP ligase YP_608111.1 (61, 76) ENW75963.1 (55, 82)
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ASA_4380 (367) AsbG, histidine decarboxylase YP_608112.1 (66, 83) ENW75964.1 (69, 84)
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33 ASA_4381 (453) TrmE, tRNA modification GTPase YP_858697.1 (98, 98)
a
Accesion number of homologues, (% amino acid identity, % amino acid similarity)
34 b
pseudogene
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4 Table 2. Description of the proteins encoded by the genes of the amonabactin cluster (loci
5 numbers correspond to the genome sequence of strain A449) in A. salmonicida and
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7 homologies to the corresponding loci in A. hydrophila.
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10 Aeromonas salmonicida subsp. salmonicida A449 Homology to Amonabactin
11 system from A. hydrophila
12 Locus (n aa) Encoded protein, description (% aa identity, % aa similarity)
13 ASA_1838 (392) EntC, Isochorismate synthase YP_856993.1 (87, 92)
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ASA_1839 (556) EntE, Siderophore synthase component E YP_856992.1 (88, 92)
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16 ASA_1840 (302) EntB, Isochorismatase YP_856991.1 (93, 95)
17 ASA_1841 (1029) AmoF, Nonribosomal peptide synthetase YP_856990.1 (91, 94)
18 ASA_1842 (259) EntA, 2,3-dihydroxybenzoate-2,3-dehydrogenase YP_856989.1 (94, 95)
19 ASA_1843 (2078) AmoG, Nonribosomal peptide synthetase YP_856988.1 (85, 89)
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ASA_1844 (536) AmoH, Nonribosomal peptide synthetase YP_856987.1 (86, 90)
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22 ASA_1845 (314) Periplasmic binding protein YP_856495.1 (94, 96)
23 ASA_1846 (392) AmoD, Phosphopantetheinyl transferase YP_856496.1 (71, 78)
24 ASA_1847 (271) ABC transporter, ATP-binding protein YP_856497.1 (93, 95)
25 ASA_1848 (351) ABC transporter, permease YP_856498.1 (92, 96)
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ASA_1849 (338) ABC transporter, permease YP_856499.1 (97, 98)
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28 ASA_1850 (657) TonB-dependent siderophore receptor YP_856500.1 (94, 98)
29 ASA_1851 (395) Major facilitator family transporter YP_856501.1 (92, 95)
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3 Table 3. Strains of A. salmonicida isolated from different hosts and PCR analysis
4 of the presence of the 43 bp deletion in amoG gene. Synthesis of acinetobactin
5 and/or amonabactin is also indicated.
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7 Intact (+) or
8 disrupted (-) Acinetobactin Amonabactin
9 Strain Isolation source amoG gene Production a Production a
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11 Isolated from fish reared in Freshwater
12 VT45.1 Brown trout + + +
13 + +
Tambre Brown trout +
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15 Xella L14,8 Brown trout + + +
16 Ulla2002 Atlantic salmon + + +
17 XellaR8G Brown trout + + +
18 Carballio vivas Brown trout + + +
19 EO 0805 Atlantic salmon + + +
20 MAO 1+1 Brown trout + + +
21 + +
Neira 18_7_09 Brown trout +
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23 Ulloa04052801 Atlantic salmon + + +
24 XellaR8P Brown trout + + +
25 X70Rin Atlantic salmon + + +
26 Ro Avia Brown trout + + +
27 007307R Atlantic salmon + + +
28 Xella L14,7 Brown trout + + +
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InvernadoiroO+ Brown trout + + +
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32 Isolated from fish reared in Seawater
33 RSP74.1 Turbot - + -
34 SF3 1/03 Turbot - + -
35 RO11.1 Turbot - + -
36 PC884.1 Turbot - + -
37 RM272.1 Turbot - + -
38 EO 0303 Atlantic salmon b - + -
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R7 190.1 Atlantic salmon b - + -
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7 Figure 1. Genetic map of the acinetobactin gene cluster and surrounding genes in A.
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10 pseudomonine gene cluster from Pseudomonas entomophila. Grey blocks link
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12 orthologous genes in each cluster. The percentage of amino acid sequence similarity in
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each protein is represented by different grey tones. The acinetobactin cluster of A.
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15 salmonicida is inserted between genes fhuE and trmE shared with A. hydrophila. DNA
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17 sequences used were those deposited in GenBank: Aeromonas hydrophila ATCC 7966
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20 Pseudomonas entomophila L48 (accesion No. NC_008027) and Acinetobacter
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22 baumannii ATCC 19606 (accesion No. AB101202).
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25 Figure 2. Structures of the siderophores identified in this study: acinetobactin (1),
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27 amonabactin P750 (2), amonabactin P693 (3), amonabactin T789 (4) and amonabactin
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T732 (5).
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32 Figure 3. 1H NMR spectra of acinetobactin (1) and amonabactin P750 (2).
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35 Figure 4. Genetic map of the amonabactin gene cluster in A. salmonicida and its
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comparison with A. hydrophila. Grey blocks link orthologous genes in each cluster. The
38 percentage of amino acid sequence similarity in each protein is represented by different
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40 grey tones. DNA sequences used were those deposited in GenBank: Aeromonas
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hydrophila ATCC 7966 (accesion No. NC_008570.1) and Aeromonas salmonicida
43 A449 (accesion No. CP000644).
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Figure 5. Growth after 18 h of incubation, DHBA production (Arnow test) and
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50 strains for genes asbD, amoG, entB and amoD and the double mutant amoGasbD. The
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52 results for complemented mutants of entB (entB/pMB290) and amoD
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3 Figure 6. Schematic pathway for the biosynthesis of siderophores acinetobactin and
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10 Figure 7. Catalytic domains prediction of the NRPS AmoG in strains A449, VT45.1
11 (produce amonabactin and acinetobactin) and RSP74.1 (produces only acinetobactin). C,
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31 comparison to that of Acinetobacter baumannii and with the pseudomonine gene cluster from Pseudomonas
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