Identification of heme uptake genes in the fish pathogen Aeromonas salmonicida subsp. salmonicida
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- Najimi, M., Lemos, M.L. & Osorio, C.R. Arch Microbiol (2008) 190: 439. doi:10.1007/s00203-008-0391-5
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Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis in fish, can use heme as the sole iron source. We applied the Fur Titration Assay to isolate a cluster including six genes hutAZXBCD that showed similarity to heme uptake genes of other Gram-negative bacteria, and three genes orf123 of unknown function. The spatial organization of these nine genes, arranged in five transcriptional units, was similar to that of a homologous cluster in A. hydrophila. When a TonB system was provided, this cluster allowed Escherichia coli 101ESD (an ent mutant, unable to synthesize enterobactin) to utilize hemin and hemoglobin as iron sources. Mutation of hutB, a gene that encodes a predicted periplasmic hemin-binding protein, caused a drastic defect in the ability of A. salmonicida to grow with hemin as unique source of iron. Interestingly, a mutant for hutA gene (encoding the outer membrane hemin receptor) showed initially a reduced ability to grow with hemin as sole iron source, but after 24 h it achieved growth levels similar to parental strain. Thus mutation of hutA could not abolish the growth with hemin as iron source, suggesting that redundant outer membrane heme transport functions might be encoded in the A. salmonicida genome.
KeywordsAeromonas salmonicidaIron uptakeHeme uptakeHemin
Bacterial pathogens have developed efficient mechanisms to obtain iron from the host, due to the lack of readily available iron in biological systems (Ratledge and Dover 2000). One of the main strategies is the synthesis and secretion of siderophores, which can remove iron from host iron-binding proteins (Crosa and Walsh 2002). Although siderophores bind iron with high affinity, they cannot remove iron from heme. Many Gram-negative pathogens have the ability to obtain iron from free heme or heme proteins by means of siderophore-independent mechanisms (Genco and Dixon 2001; Osorio and Lemos 2002). Expression of most genes required for obtaining iron, including siderophore biosynthesis and transport genes as well as heme uptake genes, are regulated by the iron-binding repressor protein Fur (ferric uptake regulator) (Braun et al. 1998).
Aeromonas salmonicida subsp. salmonicida is the causative agent of the fish disease known as furunculosis, a cause of significant economic losses in salmonid and turbot aquaculture worldwide (Toranzo et al. 2005). A. salmonicida subsp. salmonicida is known to produce siderophores for iron uptake (Chart and Trust 1983; Hirst et al. 1991; Fernandez et al. 1998), and a gene cluster encoding determinants for the biosynthesis of a catechol-type siderophore has been described recently (Najimi et al. 2008). Although it is known that certain atypical A. salmonicida strains require heme as a porphyrin source for growth in laboratory media (Ishiguro et al. 1986), to date it has not been reported whether heme compounds can be used as sole iron sources by this fish pathogen, and no heme uptake genes have been characterized at the functional level in A. salmonicida. Using a proteomic approach, a recent study demonstrated that under iron-limited conditions A. salmonicida expresses three iron-regulated outer membrane receptors, and one of these receptors was proposed to be a putative heme receptor based on sequence homology (Ebanks et al. 2004).
Heme utilization genes have been identified in numerous species, including fish pathogens as Photobacterium damselae (Rio et al. 2005), Vibrio anguillarum (Mazoy et al. 2003; Mouriño et al. 2004) and important human pathogenic bacteria as Yersinia enterocolitica (Stojiljkovic and Hantke 1992), V. cholerae (Henderson and Payne 1993; Occhino et al. 1998; Mey and Payne 2001) and Shigella dysenteriae (Mills and Payne 1997). The involvement of heme uptake in the virulence of bacterial pathogens has also been demonstrated in several species, such as V. cholerae (Henderson and Payne 1994), and Neisseria meningitidis (Stojiljkovic et al. 1995). Heme-uptake in gram negative bacteria usually involve outer membrane receptors as well as a TonB-dependent internalization process with two accessory proteins ExbB and ExbD, and this system is believed to transduce the energy of the proton motive force of the cytoplasmic membrane into transport energy required by the receptor. Subsequently, transport of heme across the cytoplasmic membrane is driven by ATP hydrolysis, and an ATP-binding cassette (ABC) transporter is involved in this transport (Köster 2001).
The goal of the present study was to identify genetic determinants involved in heme uptake in A. salmonicida subsp. salmonicida. An heme uptake cluster was sequenced and analyzed, and deletion mutants in two different genes were constructed and tested for their ability to grow with hemin as the unique source of iron. Complementation of Escherichia coli mutants with the A. salmonicida genes was performed in order to evaluate the restoration of heme utilization as an iron source.
Materials and methods
Bacterial strains, plasmids and culture conditions
Bacterial strains and plasmids used in this study
Strain or plasmid
Source or reference
RSP74.1 siderophore biosynthesis mutant (∆asbD)
Najimi et al. (2008)
MON15 hutA defective mutant
MON15 hutB defective mutant
recAthiprohsdR-M+ RP4-2-Tc:Mu-Km:Tn7 λpir
Herrero et al. (1990)
araD139 ΔlacU169rpsL150 relA1 flb5301 deoC1ptsF25rbsR aroB fhuF::λ placMu
Δ(mcrA)183Δ(mcrCB-hsdSMR-mrr)173 endA1supE44thi-1recA1gyrA96 relA1 lac
HB101 derivative, deficient in enterobactin biosynthesis Δ(entC-entA)
Crosa and Walsh (2002)
Low-copy-number cloning vector, ApR
Wang and Kushner (1991)
Cloning vector, TetR, CmR
Suicide vector derived from pCVD442, pir, ApR
Correa et al. (2000)
Cloning vector, ApR
Tabor and Richardson (1985)
Cosmid vector, ApR
FURTA-positive clone of A. salmonicida ACR168.1 containing partial heme receptor hutA and promoter region cloned into PT7-7, ApR
FURTA-positive clone of A. salmonicida ACR168.1 containing hutZ, hutX, hutB and promoter regions cloned into PT7-7, ApR
Cosmid clone from A. salmonicida ACR168.1 containing hutA-orf1-orf2-orf3-hutZX-hutBCD genes, ApR
Cosmid clone containing the heme uptake cluster of Vibrioanguillarum
Mazoy et al. (2003)
tonB-exbBD genes of Photobacterium damselae subsp. damselae cloned into pGEMT-Easy, ApR
tonB-exbBD and huvBCD genes of V. anguillarum cloned into pWKS30, ApR
Mouriño et al. (2004)
hutA-orf1-orf2-orf3-hutZX-hutBCDgenes from A. salmonicida ACR168.1 cloned into pACYC184, TetR
hutA from A. salmonicida ACR168.1 cloned into pACYC184, TetR
hutAorf1 orf2 orf3 hutZX from A. salmonicida ACR168.1 cloned into pACYC184, TetR
Recombinant DNA techniques, DNA sequencing and data analysis
Total genomic DNA was extracted and purified with the Easy-DNA kit (Invitrogen). Plasmid DNA purification and DNA extraction from agarose gels were performed with kits from QIAGEN. DNA sequences were determined by the dideoxy chain termination method using the CEQ DTCS-Quick Start Kit (Beckman Coulter) using a capillary DNA sequencer CEQ8000 (Beckman Coulter). Comparison of the sequence data with published sequences in EMBL/GenBank was performed with the blast software via the Internet (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/). Additional DNA and peptide analyses were performed using Bioedit (Hall 1999).
Fur titration assay and screening of a A. salmonicida cosmid library
Fur regulated promoters and iron-binding proteins carried on a multicopy plasmid can be identified by transformation into E. coli strain H1717, that carries a Fur-regulated fhuF::lacZ gene fusion. Fur boxes introduced on a multicopy plasmid can cause derepression of the fusion by titrating the Fur protein, and thus leading to the expression of a Lac+ phenotype. In this study we sequenced ca. 100 clones of a plasmid library of A. salmonicida ACR168.1 constructed in the indicator strain H1717 in a previous work (Najimi et al. 2008), and selected two Fur Titration Assay (FURTA)-positive clones named pFMON12 and pFMON42 that contained genes showing similarity to putative heme uptake genes in other Gram-negative bacteria.
We screened an A. salmonicida ACR168.1 cosmid library (Najimi et al. 2008) by colony-PCR using primers targeted to genes encoded in plasmids pFMON12 and pFMON42. This led to isolation of cosmid pGMON5. Cosmid DNA was purified using the QIA filter Midi Kit (QIAGEN) and used for DNA sequencing.
RNA isolation and RT-PCR
Construction of a hutA and hutB-targeted in-frame deletion mutants
Deletion mutants of the hutA and hutB genes in A. salmonicida MON15 (Table 1) were constructed by allelic exchange as previously described (Correa et al. 2000; Mouriño et al. 2004). In brief, gene deletion was carried out by using PCR amplifications of two fragments of the gene and flanking regions, which when ligated together would result in an in-frame (non polar) deletion. This process resulted in the formation of mutant alleles ΔhutA (removes coding sequence for amino acids 45-657) and ΔhutB (removes coding sequence for amino acids 107-242). The deleted alleles were cloned in the suicide vector pKEK229 (Correa et al. 2000). As a pCVD442 derivative, pKEK229 contains R6K ori, requiring the pir gene product for replication, and the sacB gene conferring sucrose sensitivity. The resulting plasmids were mated from E. coli S17-1-λpir into A. salmonicida subsp. salmonicida MON15, a derivative of strain RSP74.1 that cannot produce siderophores (Najimi et al. 2008) and transformants with the plasmid integrated in the chromosome by homologous recombination were selected on agar medium containing 50 μg ml−1 Ap (resistance conferred by pKEK229) and 20 μg ml−1 Cm (to select A. salmonicida MON15). A second recombination event was obtained by selecting for sucrose resistance (10% wt/vol) and resistance to chloramphenicol. This led to obtention of A. salmonicida MON29 (ΔhutA) and MON30 (ΔhutB) (Table 1).
Complementation of E. coli 101ESD
We tested whether the A. salmonicida heme uptake gene cluster identified after partial sequencing of cosmid pGMON5 could provide E. coli 101ESD Δ(entC-entA) with all the necessary functions to utilize hemin and hemoglobin as sole iron source. This E. coli strain lacks heme transport genes and thus constitutes an ideal heterologous host to reconstruct a heme uptake mechanism by means of providing gene combinations in recombinant plasmids. Plasmids containing different gene combinations (Fig. 1a; Tables 1, 3) were transformed in E. coli 101ESD. About 100 μl of overnight cultures of each transformant were added to 3 ml of molten soft CM9 medium and plated onto appropriate prepoured CM9 plates supplemented with 110 μM 2,2’-dipyridyl. Sterile filter-paper disks were loaded with 20 μl of either 5 mM hemin or 1 mM hemoglobin. Disks spotted with 5 mM FeSO4 were included as positive controls for utilization of iron sources. Results were annotated as positive or negative after 24 h of incubation.
Growth under iron-limiting conditions and assay methods for hemin utilization
In order to test the ability of A. salmonicida MON15 and the respective hutA and hutB defective mutants to grow in the presence of hemin as sole iron source, overnight CM9 cultures of parental and mutant strains were adjusted to an OD600 of 1, diluted 1:200 in fresh CM9 minimal medium supplemented with the iron chelator EDDA at 60 μM, and with or without addition of hemin 10 μM as sole iron source. Cultures were shaken at 22°C, and growth (OD600) was measured after 12 and 24 h.
Nucleotide sequence accession number
The EMBL accession number for the sequence described in this article is AM712659.
Results and discussion
Identification of a 9-gene cluster in A. salmonicida containing putative heme uptake genes
Proteins with homology to products of the A. salmonicida subsp. salmonicida ACR168.1 heme uptake cluster
A. salmonicida ORF (no. aa, KDa)
HutA (697, 77)
Putative heme receptor (A. salmonicida)
Putative heme receptor (A. hydrophila)
Heme receptor HuvA (Listonella anguillarum)
ORF1 (327, 35.7)
Hypothetical iron-regulated protein (A. hydrophila)
Hypothetical iron-regulated protein (Vibrio angustum)
Hypothetical iron-regulated protein (Photobacterium profundum)
ORF2 (183, 20.1)
Isochorismatase family protein (A. hydrophila)
Isochorismatase family protein (Marinomonas sp.)
Isochorismatase family protein (Erwinia carotovora)
ORF3 (142, 15.8)
Putative lipoprotein (A. hydrophila)
Putative lipoprotein (Pseudoalteromonas atlantica)
Putative lipoprotein (Shewanella baltica)
HutZ (185, 20.8)
HutZ (A. hydrophila)
Heme binding protein HutZ (V. cholerae)
HuvZ protein (L. anguillarum)
HutX (170, 19)
HuvX protein (A. hydrophila)
HuvX protein (L. anguillarum)
HugX (Plesiomonas shigelloides)
HutB (279, 29)
Periplasmic hemin transporter (A. hydrophila)
HuvB protein (L.anguillarum)
HutB (V. cholerae)
HutC (342, 35)
ABC transporter permease (A. hydrophila)
HuvC protein (L. anguillarum)
Hemin transporter, permease (V. parahaemolyticus)
HutD (262, 28.7)
ATP-binding hemin transporter (A. hydrophila)
ABC transporter, ATP binding protein HutD (V. cholerae)
ABC-type hemin transporter system, ATPase component (V. vulnificus)
Predicted protein sequences
The deduced amino acid sequences of six of the nine proteins encoded within this cluster (encoded by genes hutAZXBCD) shared significant degrees of similarity with known or predicted heme transport and heme utilization proteins in other bacteria (Table 2). The first gene of the cluster, hutA, encodes the predicted heme receptor protein of A. salmonicida. HutA is 100% identical to an A. salmonicida putative heme receptor previously sequenced in a proteome analysis (Ebanks et al. 2004), and shows similarity to a series of well-described outer membrane heme receptors.
A gene which we termed orf1 was found upstream of hutA and is transcribed from the opposite strand. It encodes a protein homologous to hypothetical iron-regulated proteins, but the involvement of this family of proteins in iron uptake remains unknown. orf2 encodes a putative isochorismatase-family protein. Isochorismatase genes are usually found clustered with genes that encode the biosynthetic machinery of catechol-type siderophores (Crosa and Walsh 2002), but are rarely associated with heme utilization genes. ORF3 showed similarity to putative lipoproteins. It is interesting to note that Reidl and Mekalanos (1996) demonstrated that a lipoprotein, e(P4), is essential for the utilization of hemin as porphyrin source in Haemophilus influenzae. However, ORF3 has no significant similarity with e(P4), and the relationship of ORF3 homologues with heme uptake is unknown.
HutZ is homologous to putative heme utilization proteins: it was suggested that Plesiomonas shigelloides HugZ could be involved in preventing heme toxicity (Henderson et al. 2001). In V. anguillarum, HutZ is essential for heme iron utilization (Mouriño et al. 2004). Similarly, V. cholerae HutZ is required for heme utilization as an iron source, and it is suggested that this protein acts as a heme storage protein as it binds heme with high efficiency (Wyckoff et al. 2004). The sixth ORF in the cluster corresponds to hutX. None of the HutX homologues has a well-known function, and although it has been suggested that Plesiomonas shigelloides HugX could be involved in preventing heme toxicity (Henderson et al. 2001), deletion of huvX in V. anguillarum showed to have no significant effect on the utilization of heme as an iron source (Mouriño et al. 2004).
The three remaining genes hutBCD encode proteins which show characteristics of heme transporters (Table 2). HutB is homologous to periplasmic heme binding proteins that transport heme across the periplasm from the receptor to the ABC transporter located in the inner membrane. HutC has homology to ABC-type permease proteins, and HutD shows homology to the ATP-binding protein component of ABC transporters involved in heme transport. HutD contains features common to ABC transporter ATPases (Walker et al. 1982), as the walkerA nucleotide binding consensus motif GPNGAGKS and an ABC transporter signature motif, LSGGE (data not shown), and presence of these features suggests that A. salmonicida HutD is the ATPase component of the heme ABC transporter.
Transcriptional organization of the heme uptake cluster
Since the inserts cloned into pFMON12 and pFMON42 yielded a positive result in the Fur titration assay, we would expect the existence of Fur binding sites (Fur boxes) upstream of the hutA gene and also in the intergenic region between hutX and hutB. As expected, 74 positions upstream of the putative ATG start codon of hutA we found a sequence (GAaAATGAgAATgATTccC) which shared 14 of 19 nucleotides with the E. coli consensus Fur box (de Lorenzo et al. 1987) (positions that match the consensus are shown in capitals). 69 positions upstream of hutB ATG start codon, another sequence (GATAATcgcttTCATTcTC) matched 13 positions out of 19 with the E. coli consensus (see Fig. 1a for location of predicted Fur boxes). The intergenic region between orf3 and hutZ shows a palindromic sequence (GCAATAAAAAACGGGCAGCCTGGGCTGCCCGTTTTTTATTGC) that constitutes a potential transcriptional terminator (Fig. 1a).
As shown in Fig. 1a, there are four genes or groups of genes which are transcribed from different orientations. This organization would suggest that there exist a minimum of four transcriptional units. To confirm this hypothesis, the cotranscription of orf123 was assayed in a reverse-transcription reaction with primer RT-1 targeted to the 3′-end of orf3, and PCR reactions were performed with primers that amplify orf1, orf2 and orf3 respectively. RT-PCR worked for orf2 (Fig. 1b), but no RT-PCR product could be obtained for orf1 when RT was carried out with primer RT-1. However, when we used primer RT-2 for reverse-transcription, PCR bands of the expected size were obtained for orf1 (Fig. 1b). This suggests that orf1 is independently transcribed, whereas orf2-orf3 are cotranscribed.
Similarly, to assay the cotranscription of hutX-hutZ on the one side, and of hutBCD on the other side, reverse-transcription was carried out with primers RT-3 and RT-4 respectively, and subsequent PCR reactions were conducted with primer pairs targeted to hutX and hutB respectively. Each RT-PCR yielded a product of the expected size (Fig. 1b), and negative and positive controls corroborated the accuracy of the RT-PCR experiment. These results altogether indicate that the nine genes of the described cluster are organized into five transcriptional units, three polycistronic ones: orf2-orf3, hutX-hutZ, hutBCD and two monocistronic ones, hutA and orf1, which would be each transcribed from their own promoters.
Similarities to other heme transport gene clusters
The A. salmonicida putative heme transport genes allow E. coli 101ESD to utilize heme and hemoglobin as sole iron sources
In order to demonstrate that the genes of this cluster play a role in heme transport, we utilized as a heterologous host the E. coli strain 101ESD. This strain lacks a genetic system for heme uptake and does not produce siderophores, and thus it cannot grow in the presence of iron chelators unless supplied with an utilizable source of iron. Genes encoding heme transport functions can thus be cloned into plasmids and tested whether they provide E. coli 101ESD with the ability to grow under conditions where heme and/or hemoglobin are the only iron sources available.
Utilization of hemin and hemoglobin as iron sources by E. coli 101ESD complemented with different combinations of A. salmonicida heme uptake genes, as well as heme uptake genes from Vibrio anguillarum and Photobacterium damselae
Hemoglobin (1 mM)
Hemin (5 mM)
FeSO4 (5 mM)
hutA-orf1-orf2-orf3-hutZX-hutBCD + tonBexbBD
hutA + huvZX tonBexbBD-huvBCD
hutAZX + tonB-exbBD
We hypothesized that one of the reasons why pMON33 did not complement 101ESD could be the inability of the E. coli TonB system to function with the A. salmonicida heme receptor. To test this possibility, we transformed 101ESD with both pMON33 and with pCAR168, a plasmid that contains the tonBexbBexbD genes from the fish pathogenic bacterium Photobacterium damselae (Table 1). Interestingly, this new gene combination allowed 101ESD to utilize both hemin and hemoglobin as iron sources, indicating that this nine-gene cluster cloned into pMON33 is functional for the transport of heme groups as long as a functional TonB system is provided.
Although no tonbexbBD genes are present within the cluster described in this study, we would expect that homologous of these genes might be located at another chromosomal location in A. salmonicida. While this study was being completed, the genome sequence of A. salmonicida A449 was made available in public databases (GenBank Accession number CP000644). An in silico search in this genomic sequence revealed the existence of genes for two different TonB systems (TonB1 and TonB2) which are not linked to the heme uptake cluster genes in that strain (data not shown).
When the A. salmonicida heme uptake cluster with the exception of hutBCD genes (pMON36, Fig. 1b) was transformed into 101ESD containing pCAR168, the transformant failed to utilize heme iron sources. This indicates that an outer membrane receptor and a TonB system genes are not sufficient for heme iron utilization, the genes encoding the predicted periplasmic and ABC-transporter genes of A. salmonicida being also necessary. Complementation of E. coli strains with heme uptake genes provided in trans to allow heme uptake has been previously reported with several genetic systems (Stojiljkovic and Hantke 1992; Mouriño et al. 2004, 2005; Rio et al. 2005). Similarly, Occhino et al. (1998) reported that utilization of hemin as an iron source can be reconstituted in E. coli 1017 (HB101 derivative, ent) with V. cholerae hutA, tonB exbBD and hutBCD genes.
We found that the 101ESD TonB system was not functional with the A. salmonicida heme transport genes. Similarly, it was reported that the Plesiomonas shigelloides heme receptor gene hugA could not complement an E. coli DHE-1 hemA mutant for the use of heme as a porphyrin source unless Plesiomonas shigelloides tonB-exbB-exbD genes were also provided in trans (Henderson et al. 2001).
Altogether, the results obtained in this section demonstrate that hutA is a functional outer membrane heme transporter, that the gene operon constituted by hutBCD is also necessary for heme uptake in E. coli 101ESD, and that this nine-gene cluster of A. salmonicida contains all the genetic determinants with the exception of a TonB system, as to allow E. coli 101ESD to use heme compounds as iron sources.
Deletion of hutA and hutB genes affects the ability of A. salmonicida to grow with hemin as sole iron source
We have demonstrated that this nine-gene cluster contains determinants for the utilization of heme groups as iron sources in E. coli 101ESD. We thus aimed at elucidating the role of these transport genes in A. salmonicida. For this purpose, in-frame deletions of hutA and hutB were constructed. We selected MON15, a RSP74.1 derivative strain, rather than ACR168.1 because this last strain is unable to grow in minimal medium, likely due to an uncharacterized auxotrophy. MON15 also harbors the heme uptake cluster described above, sharing more than 99% identity in nucleotide sequence with sequence of ACR168.1 (data not shown). In addition, MON15 is an ΔasbD mutant deficient in siderophore biosynthesis (Najimi et al. 2008), and it constitutes an ideal model to study the utilization of heme as a unique iron source because the background growth resulting from siderophore-mediated iron uptake is eliminated.
We then analyzed the effect of the hutA mutation in A. salmonicida. When the ΔhutA mutant (MON29) was grown in CM9, or in CM9 + 60 μM 2,2’-dipyridyl, it did not show significant differences with the parental strain after 12 or 24 h (Fig. 3a, b). Interestingly, when the ΔhutA strain was grown with 10 μM hemin it showed some defect in its ability to grow with this unique iron source during the first 12 h (Fig. 3a), but recovered growth levels similar to the parental strain after 24 h of culture (Fig. 3b). This result indicates that hutA is not totally essential for growth of A. salmonicida with hemin as the sole iron source, and suggests that additional outer membrane heme receptor genes exist in the genome of A. salmonicida. Presence of more than one functional outer membrane heme receptor gene coexisting in the same genome has been reported for other gram negative bacteria. This is the case of V. cholerae, where at least three functional heme receptors, namely HutA, HutR and HasR contribute to heme iron acquisition in this pathogen (Mey and Payne 2001), and mutation of any of these three receptors do not abolish completely the ability of the bacterium to use heme as iron source, being necessary the inactivation of the three genes in order to abolish hemin utilization. In V. anguillarum two different heme receptors, HuvA and HuvR have been described, although the genes for these two receptors have never been found coexisting in the same V. anguillarum strain (Mouriño et al. 2005).
An in silico search in the genome sequence of A. salmonicida A449 showed that one of the ORFs (locus ASA_2695) is annotated as an hemin receptor. Interestingly, this ORF encodes a predicted protein that shares 30% identity with the sequence of HutA described in this study, and is 37% identical to V. cholerae HutR (Mey and Payne 2001). This gene might thus encode an alternate outer membrane heme receptor. Mutational analysis of this putative heme receptor gene together with the construction of double mutants in combination with hutA, will allow a deeper insight into the genetic basis of heme transport in this important fish pathogen.
This work was supported by Grant PGIDIT06RMA26101PR-2 and Contract No. 2004/CP481, from Xunta de Galicia, Spain. Mohsen Najimi acknowledges the Ministry of Science and Education of Iran for a predoctoral fellowship.