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Applied and Environmental Microbiology, April 2008, p. 2341-2348, Vol. 74, No. 8
0099-2240/08/$08.00+0     doi:10.1128/AEM.02728-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification of Siderophore Biosynthesis Genes Essential for Growth of Aeromonas salmonicida under Iron Limitation Conditions{triangledown} ,{dagger}

Mohsen Najimi, Manuel L. Lemos, and Carlos R. Osorio*

Department of Microbiology and Parasitology, Institute of Aquaculture and Faculty of Biology, University of Santiago de Compostela, Santiago de Compostela, Galicia, Spain

Received 4 December 2007/ Accepted 13 February 2008


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ABSTRACT
 
Aeromonas salmonicida subsp. salmonicida, the etiological agent of furunculosis in fish, produces a catechol-type siderophore under iron-limiting conditions. In this study, the Fur titration assay (FURTA) was used to identify a cluster of six genes, asbG, asbF, asbD, asbC, asbB, and asbI, encoding proteins similar to components of the siderophore biosynthetic machinery in other bacteria. Reverse transcriptase PCR analyses showed that this cluster consists of four iron-regulated transcriptional units. Mutants with deletions in either asbD (encoding a multidomain nonribosomal peptide synthetase), asbG (encoding a histidine decarboxylase), or asbC (encoding a predicted histamine monooxygenase) did not grow under iron-limiting conditions and did not produce siderophores. Growth of the {Delta}asbG strain under iron starvation conditions was restored by addition of histamine, suggesting that the siderophore in this species could contain a histamine-derived moiety. None of the mutants could grow in the presence of transferrin, indicating that A. salmonicida uses the catechol-type siderophore for removal of iron from transferrin rather than relying on a receptor for this iron-binding protein. All 18 A. salmonicida strains analyzed by DNA probe hybridization were positive in tests for the presence of the asbD gene, and all of them promoted the growth of asbD, asbG, and asbC mutants, suggesting that this siderophore-mediated iron uptake system is conserved among A. salmonicida isolates. This study provides the first description of siderophore biosynthesis genes in this fish pathogen, and the results demonstrate that the asbD, asbG, and asbC genes are necessary for the production of a catecholate siderophore that is essential for the growth of A. salmonicida under iron limitation conditions.


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INTRODUCTION
 
Iron is an essential nutrient for most bacteria and serves as a cofactor in key metabolic processes. The acquisition of iron is recognized as one of the key steps for the survival of a number of bacterial pathogens within their hosts and contributes significantly to virulence (29). Thus, bacterial pathogens have developed a number of different mechanisms to obtain iron from their hosts (36). One of the main strategies to obtain iron is the synthesis and secretion of low-molecular-weight Fe(III) chelators, called siderophores, which can remove iron from host iron-binding proteins and then enter the cell through outer membrane receptor proteins. Many siderophores are small peptides synthesized by nonribosomal peptide synthetases, which are multimodular enzymes that produce peptide products with a particular sequence without an RNA template (7). Catecholate siderophores contain 2,3-dihydroxybenzoic acid (DHBA), which is synthesized from chorismate (an aromatic amino acid intermediate) through the consecutive action of a series of enzymes (35). The expression of most proteins required for siderophore biosynthesis is regulated by a global iron-binding repressor protein called Fur (ferric uptake regulator) (14).

Aeromonas salmonicida subsp. salmonicida is the etiological agent of furunculosis in fish, a disease which causes significant economic losses in cultivated salmonids in fresh and marine waters. It also affects a variety of nonsalmonid fish and has a wide distribution (34). Although the virulence mechanisms of A. salmonicida are not fully understood, several virulence factors have been reported so far; these factors include, among others, surface layer protein (5), exotoxins (20, 26), and a type III secretion system which enables the secretion and translocation of effector proteins (3, 8).

A. salmonicida is known to possess a siderophore-mediated iron uptake system, and the siderophore produced has been preliminarily characterized as a catecholate (15, 19). It has also been reported that this bacterium expresses iron-regulated outer membrane proteins when it is grown under iron-limiting conditions, and one of these proteins has been identified as a putative outer membrane ferric siderophore receptor (13). However, the exact nature of this siderophore remains unknown, and the genes involved in its biosynthesis have not been identified so far. This study was undertaken to obtain insight into the genetic basis of the siderophore-mediated iron uptake system of the fish-pathogenic bacterium A. salmonicida. The Fur titration assay (FURTA) (31) was used to identify a cluster of Fur-regulated genes involved in siderophore biosynthesis in this species, and mutational analysis of the asbD, asbG, and asbC genes demonstrated that catecholate siderophore-mediated iron uptake is an essential mechanism for the growth of this pathogen under iron-limiting conditions.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and culture conditions.
Bacterial strains and plasmids used in this study are listed in Table 1. A. salmonicida subsp. salmonicida strains were routinely grown at 22°C in tryptic soy agar and tryptic soy broth (Difco) supplemented with 1% NaCl, as well as in M9 minimal medium (24) supplemented with 0.2% Casamino Acids (Difco) (CM9). Escherichia coli strains were grown at 37°C in Luria-Bertani (LB) medium or CM9 supplemented with antibiotics when appropriate. All strains were stored frozen at –80°C in LB broth with 20% glycerol. Antibiotics were used at the following final concentrations: kanamycin, 25 µg ml–1; ampicillin (sodium salt), 50 µg ml–1; chloramphenicol, 20 µg ml–1; and gentamicin, 50 µg ml–1. All stocks were filter sterilized and stored at –20°C. The iron chelators ethylenediamine-di-o-hydroxyphenylacetic acid (EDDA) and 2,2'-dipyridyl were added to the culture medium to create iron-limiting conditions when necessary.


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  		TABLE 1. Bacterial strains and plasmids used in this study

Recombinant DNA techniques, DNA sequencing, and sequence data analysis.
Total genomic DNA was extracted and purified with an Easy-DNA kit (Invitrogen). Plasmid and cosmid DNA purification and extraction of DNA from agarose gels were performed with kits obtained from Qiagen. Southern blot analyses were performed with the ECL direct nucleic acid labeling and detection system (Amersham Biosciences) by following the manufacturer's instructions. DNA sequences were determined by the dideoxy chain termination method using a CEQ DTCS quick start kit (Beckman Coulter) and a CEQ 8000 capillary DNA sequencer (Beckman Coulter). A comparison of the sequence data with previously published sequences in the EMBL/GenBank database was performed with the BLAST software (http://ncbi.nlm.nih.gov/BLAST and http://www.ebi.ac.uk/BLAST/index.html). Prediction of protein domains was carried out by using the Pfam database online facilities (http://www.sanger.ac.uk/Software/Pfam/).

FURTA.
Fur-regulated promoters and iron-binding proteins whose genes are carried on a multicopy plasmid can be identified by transformation into E. coli strain H1717 (Table 1). This strain carries a Fur-regulated fhuF::lacZ gene fusion which is particularly sensitive to changes in the concentration of the Fur repressor. Fur boxes introduced on a multicopy plasmid can cause derepression of the fusion by titration of the Fur protein, leading to transcription of the lacZ gene and expression of a Lac+ phenotype. The FURTA was performed as previously described (31). In brief, a plasmid library of the A. salmonicida ACR168.1 genome was constructed in vector PT7-7 and transformed into E. coli H1717. Transformants positive in the FURTA were selected on MacConkey lactose agar plates containing 0.04 mM Fe2(SO4)3 and ampicillin. Plasmid DNA was isolated, and nucleotide sequences of the inserts were determined by DNA sequencing.

Construction of a cosmid library and isolation of cosmid clones containing FURTA-positive genes related to siderophore biosynthesis.
Genomic DNA from A. salmonicida ACR168.1 was partially digested with restriction enzyme Sau3AI and ligated into the SuperCos1 cosmid vector (Stratagene). Recombinant cosmids were packaged in vitro and transduced into E. coli XL1-Blue MR (Stratagene). The cosmid library was screened by performing colony PCR with pools of recombinant clones using primers targeted to genes encoding putative A. salmonicida siderophore biosynthesis genes encoded in plasmids pFMON24 and pFMON46 previously isolated using the FURTA (Table 1).

RNA purification and RT-PCR.
To determine the transcriptional organization of the siderophore biosynthesis gene cluster, A. salmonicida RSP74.1 was grown until exponential phase in low-iron CM9 (containing 40 µM EDDA), and total RNA was isolated using the RNAwiz isolation reagent (Ambion) by following the manufacturer's instructions. Reverse transcriptase PCR (RT-PCR) analyses were performed with 0.6 µg of RNA pretreated with RQ1 RNase-free DNase (Promega) by using the Moloney murine leukemia virus RT (Invitrogen). For operon mapping, a reverse transcription reaction was performed with primers RT-1 and RT-2, which are homologous to the 3' ends of asbG and asbC, respectively (Fig. 1A; see Table S1 in the supplemental material). The resulting cDNAs were used as templates for PCR amplification with Taq polymerase (Bioline), using specific primer pairs for each gene (see Table S1 in the supplemental material). A negative control PCR was performed with total RNA without Moloney murine leukemia virus RT to confirm the lack of genomic DNA contamination in each reaction mixture, and genomic DNA was used as a positive control for PCR.


Figure 1
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  		FIG. 1. (A) Physical map of the siderophore biosynthesis gene cluster of A. salmonicida subsp. salmonicida and primer design for RT-PCR. ORFs are indicated by arrows, which show the direction of transcription. RT-1, RT-2, RT-3, and RT-4 are the primers used for reverse transcription. Predicted Fur boxes identified in the intergenic regions between the asbF and asbD genes and between the asbB and asbI genes are shown, and uppercase letters indicate residues conserved in the E. coli consensus Fur box sequence. A palindromic region downstream of the stop codons of asbC and asbB is a potential transcriptional terminator. (B) Operon mapping: results of PCR amplification of the asbGFDC genes using as the template the products of RT reactions with primers RT-1 (for asbGF) and RT-2 (for asbDC). Lane M contained a molecular size marker (100 bp). The negative controls (lanes –) were PCRs without RT. The positive controls (lanes +) were PCRs in which chromosomal DNA was used as the template.

To determine the iron-mediated regulation of the transcriptional units of the cluster, RNA was purified from A. salmonicida RSP74.1 cells grown in CM9, in CM9 plus 10 µM Fe2(SO4)3, and in CM9 supplemented with 500 µM EDDA. RNA concentrations were adjusted spectrophotometrically, and RT-PCR was carried out as described above using primers RT-1, RT-2, RT-3, and RT-4 for each of the four predicted transcriptional units (Fig. 1A; see Table S1 in the supplemental material). As a control, the 16S rRNA was reverse amplified with the universally conserved oligonucleotide PH targeted to the 3' end of the 16S transcript, and PCR was carried out using universal primers internal to the 16S rRNA gene (see Table S1 in the supplemental material).

Construction of in-frame deletion mutants and gene complementation.
Internal regions of the asbG, asbC, and asbD genes in A. salmonicida RSP74.1 were deleted by the allelic exchange procedure as previously described (6) (the oligonucleotides used to amplify constructs for the mutants are described in Table S1 in the supplemental material). This process resulted in the formation of mutant alleles {Delta}asbD (which removed the coding sequence for amino acids 50 to 448), {Delta}asbG (which removed the coding sequence for amino acids 44 to 358), and {Delta}asbC (which removed the coding sequence for amino acids 28 to 388). The mutant alleles cloned in the suicide vector pKEK229 (6) were mated from E. coli S17-1-{lambda}pir into A. salmonicida RSP74.1, and cells that had undergone a double crossover were selected. This resulted in A. salmonicida strains MON15 ({Delta}asbD), MON33 ({Delta}asbG), and MON34 ({Delta}asbC) (Table 1). Southern blot hybridization analysis using suitable DNA probes was used to verify allelic exchange of the parental gene. In addition, the region involved in deletion construction was PCR amplified and sequenced to ensure that the construct was nonpolar (data not shown).

For complementation of each of the three mutants, the asbD and asbGF genes were PCR amplified along with the corresponding promoter sequences from the A. salmonicida RSP74.1 chromosome using specific primers. The asbC gene was PCR amplified from genomic DNA of the asbD mutant, so that the amplicon contained asbC and its putative promoter but not the complete asbD coding sequence. The amplified DNA fragments were cloned into plasmid pHRP309, yielding plasmids pMON38, pMON39, and pMON40 (Table 1). These plasmids were mobilized from E. coli S17-1-{lambda}pir into the corresponding A. salmonicida mutants MON15, MON33, and MON34, and transconjugants were selected on LB agar medium containing 10 µg ml–1 gentamicin and 20 µg ml–1 chloramphenicol.

Growth under iron-limiting conditions and assays for siderophore production.
The optical densities at 600 nm (OD600) of overnight LB medium cultures of parental, mutant, and complemented mutant strains were adjusted to an OD600 of 1, and the cultures were diluted 1:50 in the CM9 minimal medium and in CM9 containing the iron chelator EDDA at a concentration of 20 µM. When necessary, histamine was added to CM9 at a final concentration of 100 µM. Cultures were shaken at 22°C, and growth (OD600) was monitored after 24 h. Siderophore production was measured using the chrome azurol-S (CAS) liquid assay (30). In addition, the Arnow test (2) was used for spectrophotometric measurement of the production of DHBA (a component of catechol-type siderophores). Samples of noninoculated CM9 containing EDDA at the appropriate concentrations were used as negative controls and as spectrophotometric blanks for the CAS liquid assay.

Cross-feeding assays.
We tested the abilities of cell cultures of a collection of A. salmonicida strains (tester strains) to cross-feed the A. salmonicida mutant strains generated in this study (indicator strains). Each indicator strain was cultured on agar plates containing the CM9 minimal medium with the iron chelator 2,2'-dipyridyl at a concentration of 110 µM. Strains to be tested for production of siderophores were grown for 24 h on CM9 plates containing 110 µM 2,2'-dipyridyl. Loopfuls of cells were scrapped off and placed on top of plates previously seeded with the indicator strains. The results were considered positive when tester cells promoted the growth of indicator strains.

Utilization of iron bound to transferrin.
Apotransferrin (human) was dissolved at a concentration of 1 mM in 100 mM Tris, 150 mM NaCl, 50 mM NaHCO3 (pH 8.0) and filter sterilized. Apotransferrin was added to the medium at a final concentration of 30 µM and incubated for 60 min at 37°C prior to inoculation of cells in order to allow binding of the iron present in the medium (12). Parental and mutant strains were inoculated into the transferrin-containing medium, and growth (OD600) was monitored at 1-h intervals for 12 h.

Nucleotide sequence accession number.
The EMBL accession number for the sequences described in this paper is AM712657.


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RESULTS AND DISCUSSION
 
Isolation of a siderophore biosynthesis gene cluster using the FURTA.
In the present study two A. salmonicida subsp. salmonicida strains isolated from turbot (Scophthalmus maximus), ACR168.1 and RSP74.1 (Table 1), were assayed to determine their abilities to produce siderophores, and they were found to produce a catechol-type siderophore, yielding positive results in the CAS supernatant assay and in the Arnow chemical test (data not shown). In order to identify some of the genes involved in the biosynthesis of siderophores in these strains, we used the FURTA to screen a plasmid library of A. salmonicida ACR 168.1 for the presence of Fur-regulated promoters. Two FURTA-positive clones, pFMON24 and pFMON46 (Table 1), contained inserts (asbD and asbB) encoding proteins related to Vibrio cholerae VibF (4) and Acinetobacter baumannii BasB (23), respectively, which are proteins that have been described as part of the biosynthetic machinery of siderophores in these two species. Putative Fur boxes sharing 13 of 19 and 14 of 19 bp with the E. coli consensus Fur box (9) were located upstream of the asbD and asbB open reading frames (ORFs) (Fig. 1A). We constructed a cosmid DNA library of A. salmonicida ACR168.1 and isolated cosmid pGMON3 containing the DNA sequences of pFMON46 and pFMON24. Partial sequencing of pGMON3 revealed a ca. 11-kb fragment containing six predicted complete ORFs that was established as a putative siderophore biosynthesis gene cluster in A. salmonicida ACR168.1 (Fig. 1A).

Predicted protein sequences.
The deduced amino acid sequences of the six proteins shared significant degrees of similarity with known or predicted siderophore biosynthetic enzymes described in other bacteria, including Vibrio anguillarum and A. baumannii. The first ORF of the cluster, asbG, encodes a protein whose sequence shows high similarity to histidine decarboxylases of other bacteria, which are enzymes that catalyze the decarboxylation of histidine to histamine. These enzymes include A. baumannii BasG (68% identity) and V. anguillarum AngH (61% identity). AsbF shows high similarity with 2,3-dihydroxybenzoate-AMP ligases and isochorismatases, which are enzymes involved in the synthesis and/or activation of DHBA, a component of catechol-type siderophores. The closest relatives of AsbF include A. baumannii BasF (55% identity) and E. coli EntB (34% identity).

asbD encodes a protein that shows 61, 54, and 47% identity with the nonribosomal peptide synthetases BasA/D, VibF, and BasD of Pseudomonas entomophila, A. baumannii, and V. cholerae, respectively. A domain prediction analysis of AsbD using the Pfam database showed that AsbD contains domains typical of nonribosomal peptide synthetases involved in siderophore biosynthesis in bacteria (data not shown). Similarly, AsbB shows identity to the nonribosomal peptide synthetases V. anguillarum AngM (10) (41% identity) and A. baumannii BasB (23) (49% identity), and comparison of AsbB with the Pfam database showed that it has a structure typical of nonribosomal peptide synthetases (data not shown).

The protein encoded by the fourth ORF, asbC, shows high similarity to enzymes involved in the synthesis of siderophores containing histamine or derivatives of histamine in their structures, such as A. baumannii BasC (70% identity) and V. anguillarum AngU (63% identity). The last ORF, asbI, encodes a protein that shows 37% identity with E. coli FecI, a sigma factor belonging to the extracytoplasmic function family. Members of this family have been described as regulatory components of iron transport systems in other bacterial species (1, 21).

Upstream of asbG we found genes encoding a putative tRNA modification GTPase, as well as a series of hypothetical proteins. Downstream of asbI we sequenced two genes with homology to genes encoding transporters belonging to the ABC family (data not shown), which could be involved in the internalization of the Fe-siderophore complex. In other bacterial species, ABC transporter genes involved in the internalization of the siderophore have been found in the vicinity of the biosynthetic genes (7). No additional genes with homology to enzymes potentially involved in siderophore biosynthesis were found in the vicinity of the six-gene cluster described in this study.

Transcriptional organization and iron regulation of the six-gene cluster.
The genetic organization of the cluster shows that asbD and asbC are transcribed from the strand opposite the strand containing asbG and asbF, whereas asbB and asbI are divergently transcribed. In addition, two regions showing similarity to the Fur box consensus were found in intergenic regions between the asbF and asbD genes and between the asbB and asbI genes, and a putative transcriptional terminator was found downstream of the asbC and asbB genes (Fig. 1A). These data suggest that this cluster is organized in four transcriptional units: asbG-asbF, asbD-asbC, asbB, and asbI. To confirm this, RT-PCR analyses were carried out with A. salmonicida RSP74.1 total RNA. When the cDNA obtained using primer RT-1 for reverse transcription was used as the PCR template, PCR products whose sizes corresponded to the sizes predicted for the asbG and asbF genes were amplified (Fig. 1B), indicating that the asbG and asbF genes are cotranscribed. Similarly, using reverse transcription primer RT-2, we confirmed the presence of a polycistronic mRNA comprising asbD and asbC which would have been cotranscribed from the promoter located upstream of asbD (Fig. 1B).

The expression of RNA transcripts of the four transcriptional units was analyzed under normal, iron-rich, and iron-limiting conditions using reverse transcription with primers RT-1, RT-2, RT-3, and RT-4. The results showed that the four transcripts were weakly expressed under iron-rich conditions; the intensities of the bands were significantly reduced compared to the intensities of the bands obtained from samples grown under iron limitation conditions (Fig. 2). The level of 16S RNA expression was not significantly affected by the iron conditions of the medium. These results indicate that the six-gene cluster is iron regulated, and the presence of conserved Fur boxes suggests that Fur is responsible for this iron-mediated regulation. However, additional studies are needed to demonstrate that Fur is indeed involved in the iron regulation of this cluster.


Figure 2
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  		FIG. 2. RT-PCR detection of mRNA of A. salmonicida RSP74.1 siderophore biosynthesis genes under different iron conditions. Lanes 1, CM9 with 10 µM FeSO4; lanes 2, CM9; lanes 3, CM9 with 500 µM EDDA. The amplified fragments in the genes (representing each of the four transcriptional units of the cluster) are as follows: asbG, 480-bp fragment; asbD, 283-bp fragment; asbB, 312-bp fragment; and asbI, 328-bp fragment. Reverse transcription was carried out with the following primers (shown in Fig. 1A): RT-1 for asbG, RT-2 for asbD, RT-3 for asbB, and RT-4 for asbI. As a control, a 189-bp fragment of the 16S rRNA gene was amplified after previous reverse transcription with primer PH (see Table S1 in the supplemental material).

asbG and asbC are essential for growth under iron limitation conditions and are related to biosynthesis of histamine and derivatives of histamine.
A mutant with an in-frame mutation in the asbG gene (encoding a predicted histidine decarboxylase) was constructed, and its ability to grow under iron limitation conditions and to produce siderophores was tested. We selected A. salmonicida strain RSP74.1 for this analysis rather than strain ACR168.1. RSP74.1 also harbors the siderophore biosynthesis cluster described above (data not shown) and is able to grow in the CM9 minimal medium, whereas strain ACR168.1 was found to be unable to grow in this minimal medium, probably because of an uncharacterized auxotrophy. When the MON33 mutant ({Delta}asbG) was cultured in the CM9 minimal medium, no significant differences in the levels of growth were observed compared with the RSP74.1 parental strain. However, the growth of MON33 was impaired under iron-restricted conditions (CM9 plus 20 µM EDDA) (Fig. 3), indicating that asbG is essential for growth under iron limitation conditions. The mutant complemented with the asbG gene in plasmid pMON39 was able to grow in CM9 plus 20 µM EDDA at levels similar to those of the parental strain (Fig. 3). If asbG plays a role in providing a histamine derivative for biosynthesis of the siderophore, we would predict that a mutation in this gene would not prevent DHBA production, while siderophore assembly should in turn be eliminated. As expected, the CAS test showed that there was a >2-fold decrease in the level of siderophore activity, whereas the level of DHBA production remained similar to the wild-type level (Fig. 4). The residual activity in the CAS assay observed for the mutant strain can be attributed to the residual iron-chelating activity of incomplete siderophore molecules, as reported previously for other catecholate siderophores (17).


Figure 3
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  		FIG. 3. Growth (OD600) after 24 h of incubation of the A. salmonicida RSP74.1 parental strain, the asbD, asbG, and asbC mutants, and these mutants complemented with plasmids pMON38, pMON39, and pMON40, respectively. The conditions were as follows: CM9, CM9 plus EDDA 20 µM (iron-deficient conditions), CM9 plus 20 µM EDDA supplemented with 100 µM histamine (Hist), and complemented mutants grown in CM9 plus 20 µM EDDA (iron-deficient conditions). The data are the means from three independent experiments, and the error bars indicate standard errors.


Figure 4
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  		FIG. 4. Siderophore production measured by the CAS supernatant assay (A630) (grey bars) and DHBA production measured by the Arnow test (A510) (cross-hatched bars). In the CAS assay, lower values indicated higher siderophore production. The data are the means from three independent experiments.

As histamine is the final product of the reaction catalyzed by histidine decarboxylase, we examined whether addition of this compound could complement the inability of MON33 to grow under iron limitation conditions. As shown in Fig. 3, after 24 h of growth in the presence of 100 µM histamine, the cell density values for the asbG mutant were similar to those for the parental strain. Similarly, a paper disk containing 20 µl of 10 mM histamine placed on top of a CM9 agar plate on which the asbG mutant had previously been seeded promoted the growth of the asbG mutant (data not shown). All the results described here indicate that the product of asbG plays a crucial role in the biosynthesis of the A. salmonicida siderophore and that histamine is a precursor in siderophore biosynthesis. In V. anguillarum histamine is a precursor of the siderophore anguibactin, and a histidine decarboxylase gene was found to be essential for its biosynthesis (33). Similarly, pseudomonine from Pseudomonas fluorescens AH2 (2) and acinetobactin from A. baumannii (38) also have histamine-derived moieties in their structures, and the proteins involved in histidine decarboxylation in these species also show similarity to A. salmonicida AsbG.

We then studied the role of AsbC in siderophore synthesis in A. salmonicida RSP74.1. When an asbC deletion mutant (strain MON34) was cultured in CM9, no significant differences in the levels of growth were observed compared with the parental strain (Fig. 3). However, the ability of MON34 to grow under iron-restricted conditions (CM9 plus 20 µM EDDA) was severely affected, and addition of histamine to the culture medium did not enhance the growth of the mutant (Fig. 3). MON34 was able to grow in iron-limiting conditions when it was complemented with plasmid pMON40 containing the asbC gene (Fig. 3). Chemical tests showed that siderophore production, but not DHBA production, was significantly reduced (>2-fold decrease) in the asbC mutant (Fig. 4). Altogether, these results demonstrate that asbC is essential for growth of A. salmonicida RSP74.1 under iron-limiting conditions and that AsbC is part of the siderophore biosynthesis pathway.

AsbC shows similarity to A. baumannii BasC (23), Sinorhizobium meliloti RhbE (22), and V. anguillarum AngU (10). RhbE has been described as a 1,3-diaminopropane N-hydroxylase and might be involved in the oxidation of histamine. BasC is predicted to be involved in the oxidation of histamine, yielding N-hydroxyhistamine, one of the constituents of acinetobactin. AngU is a putative monooxygenase presumed to be involved, together with AngH (a homologue of AsbG), in the biosynthesis of anguibactin by catalyzing the production of N-hydroxyhistamine from histidine (7). Taken together, these observations suggest that AsbC is a putative histamine monooxygenase and that the A. salmonicida siderophore might have N-hydroxyhistamine or another derivative in its structure.

asbD and asbB encode nonribosomal peptide synthetases: analysis of an asbD mutant.
The AsbD and AsbB proteins contain domains typical of nonribosomal peptide synthetases (see above) and show identity to different domains of VibF, a well-described multifunctional nonribosomal peptide synthetase involved in vibriobactin biosynthesis in V. cholerae that assembles vibriobactin from the three precursors 2,3-DHBA, L-threonine, and norspermidine (4). In order to investigate whether the A. salmonicida siderophore is synthesized using a nonribosomal peptide synthetase mechanism, we used the asbD gene to construct a defective mutant by allelic exchange (MON15) (Table 1). When the mutant and parental strain were cultured in the CM9 minimal medium, no significant differences in the levels of growth of these two strains were observed (Fig. 3). However, under iron-restricted conditions (CM9 plus 20 µM EDDA), the ability of MON15 to grow was severely affected compared with the parental strain. Complementation of the MON15 mutant with the asbD gene provided in plasmid pMON38 restored the growth to almost wild-type levels (Fig. 3). The production of DHBA in the mutant was similar to that in the parental strain (Fig. 4). However, analysis of the supernatants by the CAS assay revealed that siderophore production was significantly reduced in mutant strain MON15; the absorbance in the CAS assay was ca. threefold lower than that for the parental strain (Fig. 4). Mutation of asbD would be expected to eliminate the assembly of the complete siderophore molecule but not the synthesis of DHBA, since domain analyses using the Pfam database did not predict domains for DHBA synthesis or activation in AsbD.

These results suggest that AsbD and AsbB are members of the family of nonribosomal peptide synthetases involved in synthesis of the A. salmonicida siderophore and demonstrate that the function encoded by asbD is essential for siderophore biosynthesis and for the growth of A. salmonicida under iron limitation conditions. The sequence identity of A. salmonicida AsbD and AsbB with A. baumannii BasD and BasB, respectively, and with V. anguillarum AngN and AngM, respectively, is interesting. It has been reported recently that the siderophore synthesis systems of some A. baumannii and V. anguillarum strains are structurally and functionally related (11). It is tempting to speculate that the siderophore produced by A. salmonicida could also be structurally and functionally related to anguibactin and/or acinetobactin. Experiments to examine this possibility will be the subject of future studies.

A. salmonicida siderophore is essential for utilization of iron bound to transferrin.
Most iron in the vertebrate hosts is bound to iron-binding proteins, including transferrin, or is sequestered intracellularly. In order for bacteria to grow and infect an animal host, they must first take up iron effectively. We thus attempted to ascertain whether the catecholate siderophore produced by A. salmonicida allows this important fish pathogen to utilize transferrin-bound iron. For this purpose, we tested the parent and the three mutants constructed in this study to determine their abilities to grow in CM9 with 30 µM apotransferrin, in which most of the iron is expected to be bound to the protein. We found that the parental strain RSP74.1 could grow at acceptable levels in the presence of holotransferrin. However, the growth of the asbG, asbC, and asbD mutants was impaired under these conditions (Fig. 5). Interestingly, addition of 100 µM histamine to the asbG mutant partially enhanced growth in the presence of 30 µM transferrin. Altogether, these results indicate that the siderophore produced by A. salmonicida is required for iron assimilation from transferrin and show that A. salmonicida uses the catechol-type siderophore for removal of iron from transferrin rather than relying on a receptor for this iron-binding protein. Other pathogens capture transferrin-bound iron by means of siderophore-independent mechanisms and possess specific outer membrane receptors that recognize holotransferrin, as is the case in Haemophilus and Neisseria species (29).


Figure 5
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  		FIG. 5. Growth of A. salmonicida strains in CM9 supplemented with 30 µM transferrin. {blacksquare}, parental strain RSP74.1; {triangleup}, {Delta}asbG strain; {circ}, {Delta}asbD strain; {diamond}, {Delta}asbC strain; {blacktriangleup}, {Delta}asbG strain with 100 µM histamine added. The data are means from three independent experiments.

Distribution of the asbD gene in A. salmonicida strains and cross-feeding assays.
The presence of genes of the siderophore synthesis cluster described in this study in a collection of A. salmonicida strains that had different geographical origins and were isolated from salmonids and turbot (Table 1) was tested by Southern hybridization. The asbD gene was found to be present in all the assayed strains, and it yielded bands that were similar sizes in all the cases (data not shown). This suggests that the siderophore biosynthesis genes are conserved in A. salmonicida. In other bacterial fish pathogens different siderophore biosynthesis systems have been reported to show strain specificity. This is true for V. anguillarum (10) (in which at least one of the siderophore synthesis systems is plasmid encoded) and Photobacterium damselae subsp. piscicida (27). However, we have evidence that the A. salmonicida gene cluster described in the present study is located on the chromosome, since the plasmid profiles of the strains analyzed have been shown to be different depending on the isolation source and there are no high-molecular-weight plasmids that are present in all the strains (25).

We further tested whether a collection of A. salmonicida strains isolated from turbot and from salmon (Table 1) could cross-feed the asbD, asbG, and asbC mutants constructed in this study. We found that all 18 A. salmonicida isolates tested were able to promote the growth of each of the three mutants under iron limitation conditions. This observation strongly suggests that all the A. salmonicida strains used in this study produce a siderophore that can be efficiently used by the mutant strains. In a previous study, Fernandez et al. (15) analyzed 17 A. salmonicida strains isolated from salmonids in Spain and Scotland and found that all of them produced a catecholate siderophore. Moreover, culture supernatants of these strains stimulated the growth of all the homologous isolates and most of the heterologous isolates. These observations led these authors to propose that A. salmonicida appears to be a homogeneous species with respect to siderophore production (15). The results obtained in the present study clearly support this previous proposal.

Conclusions.
We successfully used the FURTA to identify a gene cluster involved in siderophore production in A. salmonicida. Three assayed genes, asbG, asbC, and asbD, were demonstrated to play a crucial role in the biosynthesis of the A. salmonicida siderophore. Although the chemical structure of this siderophore remains unknown, our results indicate that it is a catechol-type siderophore, is synthesized by nonribosomal peptide synthetases, and might contain a histamine-derived moiety. Interestingly, we demonstrated that the iron uptake mechanism based on production of siderophores is crucial for the growth of A. salmonicida under iron limitation conditions and is necessary for the utilization of iron bound to transferrin. This strict dependence suggests that siderophore-mediated iron uptake must be an important virulence factor in this fish pathogen. Studies of the role of this system in the virulence of A. salmonicida for fish are under way.


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ACKNOWLEDGMENTS
 
This work was supported by grant PGIDIT06RMA26101PR-2 and contract 2004/CP481 from Xunta de Galicia, Spain. M.N. acknowledges the Ministry of Science and Education of the IR of Iran for a predoctoral fellowship.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Parasitology, Institute of Aquaculture and Faculty of Biology, University of Santiago de Compostela, 15782 Santiago de Compostela, Galicia, Spain. Phone: (34) 981563100, ext. 16062. Fax: (34) 981547165. E-mail: crosorio{at}usc.es Back

{triangledown} Published ahead of print on 22 February 2008. Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back


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Applied and Environmental Microbiology, April 2008, p. 2341-2348, Vol. 74, No. 8
0099-2240/08/$08.00+0     doi:10.1128/AEM.02728-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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