Vibrio anguillarum can utilize hemin and hemoglobin as sole iron sources . In previous work we identified HuvA, the V . anguillarum outer membrane heme receptor by complementation of a heme utilization mutant with a cosmid clone [pML1] isolated from a genomic library of V . anguillarum . In the present study, we describe a gene cluster contained in cosmid pML1, coding for nine potential heme uptake and utilization proteins: HuvA, the heme receptor;HuvZ and HuvX; TonB, ExbB, and ExbD; HuvB, the putative periplasmic binding protein; HuvC, the putative inner membrane permease;and HuvD, the putative ABC transporter ATPase . A V . anguillarum strain with an in-frame chromosomal deletion of the nine-genecluster was impaired for growth with heme or hemoglobin as thesole iron source . Single-gene in-frame deletions were constructed, demonstrating that each of the huvAZBCD genes are essential for utilization of heme as an iron source in V . anguillarum, whereas huvX is not . When expressed in Escherichia coli hemA [strain EB53], a plasmid carrying the gene for the heme receptor, HuvA, was sufficient to allow the use of heme as the porphyrin source . For utilization of heme as an iron source in E . colient [strain 101ESD], the tonB exbBD and huvBCD genes were required in addition to huvA . The V . anguillarum heme uptake cluster shows some differences in gene arrangement when compared to homologous clusters described for other Vibrio species.

 
Iron is an essential element for most bacteria, serving as acofactor in key metabolic processes such as nucleotide biosynthesis,electron transfer, and energy transduction . Most bacterial pathogensrequire iron for growth and to establish an infection, and thusthey have developed efficient mechanisms to obtain iron fromthe host [32] . The small amounts of extracellular iron are quicklybound by high-affinity carrier proteins such as transferrinin serum and lactoferrin in secretions . Vibrio anguillarum isthe etiological agent of a septicemic disease known as vibriosis,which affects a large number of marine fish species . Withinthe 10 O serogroups described by Sørensen and Larsen[35], only serotypes O1 and O2 and, to a lesser extent, serotypeO3 are considered important fish pathogens . Although V . anguillarum is the best known fish pathogen of the genus Vibrio, the nature of its virulence mechanisms is not thoroughly understood . Strains of pathogenic V . anguillarum serotypes can acquire iron by the production and secretion of siderophores [2, 4, 16, 19, 38].Heme and some heme-containing proteins, including hemoglobinand hemoglobin-haptoglobin, can also be used as iron sourcesby a siderophore-independent mechanism [23, 24].

In this way, many gram-negative pathogens have the ability to obtain iron through utilization of free heme or heme proteinsfrom the host tissues [7, 18], and heme utilization genes havebeen identified in numerous species, including Yersinia enterocolitica[36, 37], Vibrio cholerae [13, 26, 29], Escherichia coli O157[39], Vibrio vulnificus [20], Plesiomonas shigelloides [14],and Shigella dysenteriae [28] among others . Specific receptorsare involved in heme binding and transport . Receptor-mediateduptake of heme includes translocation of the ligands into theperiplasm by an energy-dependent process that requires a functionalTonB system [30, 36] . The TonB protein, which is anchored inthe cytoplasmic membrane and associated with two accessory proteins,ExbB and ExbD, spans the periplasm and interacts with the ligand-loaded receptor . The TonB-ExbBD system is believed to be involved in transducing the energy of the proton motive force of the cytoplasmic membrane into transport energy required by the receptor . The pernicious oxidative effects of free heme dictate the presenceof a periplasmic binding protein to transport heme across theperiplasmic compartment . Transport of heme or iron across thecytoplasmic membrane is driven by ATP hydrolysis, and an ATP-bindingcassette [ABC] transporter is commonly involved in transportthrough the cytoplasmic membrane [17].

In previous studies we identified HuvA, the outer membrane receptor involved in heme uptake in V . anguillarum . The huvA gene was isolated from a V . anguillarum H-775-3 cosmid library by its ability to restore heme utilization in 101ESD, an E . coli mutant strain that fails to grow under iron-limiting conditions and cannot use heme as an iron source [25] . A TonB-ExbB-ExbD systemhas also been recently found in V . anguillarum . It was observedthat a tonB mutant strain was still able to take up heme, suggestingthat V . anguillarum may harbor two TonB systems [M . Stork, M.Di Lorenzo, S . Mouriño, C . R . Osorio, M . L . Lemos, andJ . H . Crosa, unpublished data].

The goal of the present study was to identify and characterizethe genes involved in heme transport in V . anguillarum . Sequences of the heme uptake cluster genes were determined and analyzed,and deletion mutants were constructed and tested for the abilityto grow with hemin or hemoglobin as the sole iron source . Complementationof E . coli mutants with V . anguillarum genes for restoration of heme utilization as an iron and porphyrin source was also evaluated.

 
Plasmids, bacteria, and media. Plasmids and bacterial strains used in this study are listedin Table 1 . V . anguillarum cells were routinely grown at 25°Cin tryptic soy agar [Difco] supplemented with 1% NaCl [TSA-1],as well as in M9 minimal medium [27] supplemented with 0.2% Casamino Acids [Difco] [CM9] . E . coli strains were grown at 37°C in Luria-Bertani [LB] broth, LB agar, or CM9 supplementedwith antibiotics when appropriate . Strain EB53 aroB hemA andits derivatives were routinely grown in LB medium supplementedwith 2 µg of 5-aminolevulinic acid [ALA; Sigma] ml^–1.All strains were stored frozen at –80°C in LB brothwith 20% glycerol . Antibiotics were used at the following finalconcentrations: tetracycline hydrochloride at 15 µg ml^–1,kanamycin at 25 µg ml^–1, and ampicillin sodium saltat 50 µg ml^–1 . 2,2'-Dipyridyl [Sigma], used to chelatenonheme iron, was prepared at 10 mM in ultrapure water [milli-Q;Millipore] . Bovine hemin [Sigma] was dissolved at 5 mM in 10mM NaOH . Bovine hemoglobin [Sigma] was dissolved at 1 mM inultrapure water.

 
Recombinant DNA techniques. Recombinant DNA methods including restriction-enzyme digestions,ligation reactions, agarose gel electrophoresis, and plasmidanalysis were performed following standard protocols [34] . ChromosomalDNA was isolated by using the Easy-DNA kit [Invitrogen] . Plasmidpurification and elution of DNA fragments from agarose gelswere performed with kits from QIAGEN . Southern blot analyseswere performed with Hybond-N+ membranes [Amersham Biosciences],using the ECL Direct Nucleic Acid Labeling and Detection system[Amersham Biosciences], following the manufacturer's instructions.E . coli strains were transformed by a standard calcium chloridemethod [34] . Triparental mating for transfer of cosmid pML1into E . coli EB53 was performed as described previously [8]. E . coli strain HB101 harboring the helper plasmid pRK2013 was used as a mobilizing strain [5].

DNA sequencing and data analysis. DNA sequences were determined by the dideoxy-chain terminationmethod by using the Big Dye Terminator v3.0 DNA sequencing kit[Applied Biosystems] on an automated sequencer, ABI 377 [AppliedBiosystems] . Restriction maps were generated and DNA translationwas performed with the BioEdit Sequence Alignment Editor [11].Homologies of the deduced amino acid sequences were determinedby consulting the EMBL and SWALL databases, using the FASTA3and BLAST algorithms, at the European Bioinformatics Institutewebsite.

Construction of chromosomal mutations. Gene deletions in V . anguillarum H-775-3 were constructed byusing PCR amplifications of two fragments of each gene, whichwhen ligated together would result in an in-frame [nonpolar]deletion . Amplification was carried out using the Expand HighFidelity PCR system [Roche] . The oligonucleotides used to amplifythe carboxy- and amino-terminal fragments of each gene are listedin Table 2 . Construction of an in-frame huvAZXBCD tonB exbBD mutant was accomplished by ligating the PCR products obtained with primer pairs HuvA.A-HuvA.B and HuvD.C2-HuvD.D . Allelicexchange was carried out using the suicide vector pNidKan . PlasmidpKEK229 [3] was cut with PstI, and a kanamycin resistance cassette[Genblock; Pharmacia] was inserted, producing pNidKan . As apCVD442 derivative, pNidKan contains R6K ori, requiring thepir gene product for replication, and the sacB gene, conferringsucrose sensitivity . Construction of in-frame deletions of huvX, huvZ, huvC, huvD, huvB, huvA and deletion of the complete genecluster occurred in several steps . The PCR-amplified carboxy-terminalgene fragments were ligated into pWKS30, and resulting plasmidswere cut with appropriate enzymes and ligated to the amino-terminalPCR fragments of each corresponding gene . This process resultedin the formation of mutant alleles huvZ [removes coding sequencesfor amino acids 76 to 126], huvX [removes coding sequences foramino acids 90 to 144], huvC [removes coding sequences for aminoacids 19 to 291], huvB [removes coding sequences for amino acids18 to 259], huvD [removes coding sequences for amino acids 183to 345], and huvA [removes coding sequences for amino acids178 to 698] and the complete gene cluster deletion mutant huv [removes coding sequences encompassing amino acid 178 encoded by huvA and amino acid 345 encoded by huvD] . Each deleted allele cloned in pWKS30 was digested with NotI and SalI and ligated into the NotI/SalI sites of the suicide vector pNidKan . Theresulting plasmids were mated from E . coli S17-1-pir into V.anguillarum H-775-3, and transformants with the plasmid integratedinto the chromosome by homologous recombination were selectedon agar medium containing kanamycin and ampicillin . A secondrecombination event was obtained by selecting for sucrose resistance[10% wt/vol] and resistance to the specific antibiotic for therecipient strain [ampicillin] . This led to obtention of V . anguillarum huvZ, huvX, huvC, huvB, huvD, huvA, and huv mutant strains.Southern blot hybridization analysis was used to verify allelicexchange of the parental gene . In addition, for each mutantstrain, the region involved in the deletion construction was PCR amplified and sequenced to ensure that the constructs were nonpolar [data not shown].

 
Subcloning of pML1 and construction of plasmids carrying V . anguillarum genes. Oligonucleotides used to amplify genes of the V . anguillarumheme uptake system are listed in Table 2 . Cosmid pML1 was cutwith ApaLI, and two fragments were subcloned in pACYC177, yieldingpCAR121 [containing huvA] and pCAR126 [containing huvX, tonB-exbBD, huvB, and partial huvZC genes] . An EcoRV fragment containing complete huvZX genes was cloned in pWKS30 to yield pSML11 . A SacI-HindIII fragment including complete tonB-exbBD genes was cloned in pWKS30 to yield pSML34.

The complete nine-gene heme uptake cluster was PCR amplifiedwith primers huvA-5' and HuvC.D . The PCR product was cut withEcoRI and cloned in pWKS30 to produce pSML23 . A PstI-EcoRI fragmentof pSML23 containing huvBCD genes, and a SacI-ApaI fragment,including the tonB system plus huvBCD genes, were subclonedin pWKS30 to yield pSML32 and pSML33, respectively [in caseswhere a unique promoter exists upstream of tonB from which thesix downstream genes are transcribed, huvBCD genes cloned in pSML32 will not be expressed].

The complete huvA gene including upstream and downstream DNA was amplified with primers huvA-5' and huvA-Eco-3' . The PCR product was cut with EcoRI and cloned in pWKS30 to yield pCAR115.The complete huvX gene including upstream DNA was amplifiedwith primers VA-huvZ-D and VA-HutZ-1 . The PCR product was clonedin pGEMT-Easy, and the insert was cut with EcoRI and subclonedin pWKS30 to yield pCAR181 . The complete huvZ gene includingthe putative huvX promoter and in-frame-deleted huvX open reading frame [ORF] was amplified from V . anguillarum huvX with primersPX3 and VA-TonB-4 [this allows the cloning of huvZ independentlyof huvX while still maintaining the putative huvX promoter,from which it is feasible that transcription of huvZ may occur].The PCR product was cloned in pGEMT-Easy, and the insert wascut with EcoRI and cloned in pWKS30 to yield pCAR179 . All thePCR-amplified V . anguillarum genes to be used in complementationassays were obtained with the Expand High Fidelity PCR system[Roche] and further DNA sequenced to ensure that no PCR errorswere artificially introduced.

Hemin and hemoglobin utilization assays. To test the ability of V . anguillarum mutants to utilize heminor hemoglobin as an iron source, overnight cultures of the parentalstrain, V . anguillarum H-775-3, and the mutant strains wereadjusted to the same optical density and diluted 1:100 in freshCM9 broth containing the iron source [hemin, 10 µM; orhemoglobin, 1 µM] with or without the iron chelator 2,2'-dipyridylat 100 µM . Cultures were shaken at 25°C, and absorbanceat 600 nm was monitored at 1-h intervals over 12 h.

Complementation experiments. To test which genes of the cluster were essential for the utilizationof hemin and hemoglobin as iron and porphyrin sources, E . colistrain EB53 aroB hemA and strain 101ESD [entC-entA] were transformedwith several plasmids . One-hundred-microliter portions of overnightcultures were added to 3 ml of molten soft nutrient broth [NB]or CM9 and plated onto appropriately prepared NB, CM9, or CM9supplemented with 100 µM 2,2'-dipyridyl plates . Sterile filter paper disks were loaded with 20 µl of 5 and 0.05mM hemin and 1 mM hemoglobin . Disks spotted with 20 µlof 2 mg-ml^–1 ALA or 5 mM FeSO₄ were included as positivecontrols for utilization of porphyrin and iron sources, respectively.Results were annotated as positive or negative after 24 h ofincubation.

Nucleotide sequence accession number. The EMBL accession number for the sequence described in thisarticle is AJ496544.

 
Nucleotide sequence analysis of V . anguillarum heme utilization genes. The genetic organization of the V . anguillarum heme utilizationgene cluster was determined by partial DNA sequence analysisof pML1, a cosmid which enabled E . coli 101ESD to utilize hemeas an iron source and which contains the heme receptor gene,huvA [25] . The two strands of a DNA region downstream of huvAspanning ca . 6,000 bp were sequenced, and eight closely linkedORFs were identified [Fig. 1] . The predicted products displaysignificant similarity to components of other heme uptake systemsin Vibrio and Plesiomonas species [Table 3].

 
A gene which we termed huvZ was found 125 bp downstream of the huvA stop codon and is transcribed from the opposite strand. huvZ encodes a 176-amino-acid protein with homology to proteins linked to heme transport systems [Table 3] . The function ofHuvZ homologues remains uncharacterized, though some observations suggest that P . shigelloides HugZ could be involved in preventing heme toxicity [14] . Interestingly, database comparisons evidencethat HuvZ has homology to proteins containing a flavin mononucleotide-bindingsplit barrel [data not shown] . This homology suggests that thisprotein may play a role in processes of electron transfer, withthe heme group being involved.

The next ORF in the cluster corresponds to the huvX gene, located 55 nucleotides upstream of the huvZ start codon, and codes for a predicted protein of 171 amino acids . The highest similarities were to proteins linked to heme transport systems of gram-negative bacteria [Table 3] . None of the HuvX homologues has a knownfunction, but it has been suggested that P . shigelloides HugXcould be involved in preventing heme toxicity [14].

Six additional genes are transcribed from the same strand as huvA . The three ORFs located adjacent to huvX encode the three proteins TonB, ExbB, and ExbD . The remaining three ORFs located downstream of exbD in this cluster code for proteins which show characteristics of heme transport proteins [Table 3] . The predictedstart codon for the seventh ORF of the cluster, huvB, is 51nucleotides downstream from the stop codon of exbD . It encodesa predicted 282-amino-acid protein with homology to putative periplasmic heme-binding proteins . These proteins are believed to be involved in the transport of heme across the periplasmfrom the receptor to the ABC transporter located in the innermembrane.

The eighth ORF of the cluster encodes a 314-amino-acid protein which we termed HuvC, which has homology to members of a familyof ABC-type permease proteins involved in the uptake of iron[Table 3] . The last ORF of the cluster encoded a protein termed HuvD, which showed homology to the ATP-binding protein component of permeases involved in heme transport [Table 3] . The firstputative ATG of the V . anguillarum HuvD ORF determines a proteinof 199 amino acids, which is shorter than homologues such as V . cholerae HutD or P . shigelloides HugD . Two TTG triplets, which is an unusual start codon in eubacteria [9], are locatedin frame upstream of the candidate ATG start codon and can be considered putative start codons for V . anguillarum HuvD . The amino acid sequence translated from any of these TTG triplets show high similarity to the N-terminal amino acids of V . cholerae HutD . Considering one of these two in-frame TTG triplets asthe start codon, the larger V . anguillarum predicted HuvD ORF encodes a protein with features common to ABC transporter ATPases: the walkerA nucleotide-binding consensus motif GPNGAGKS [located42 bp upstream of the first ATG codon] and the walkerB motifLMLDE at positions 103 to 107 downstream of the putative ATGstart . These two sites are part of a highly conserved ATP-bindingmotif that constitute an ATP-binding pocket [40] . In addition, an ABC transporter signature motif, LSGGE, is found at positions 77 to 81 of HuvD [Fig . 2] . The presence of these features suggeststhat V . anguillarum HuvD is the ATPase component of the hemeABC transporter.

 
Similarities with other heme uptake gene clusters. The spatial organization of heme uptake genes in the chromosomeof V . anguillarum is similar to that described for other Vibrio species and for P . shigelloides [Fig . 3] . This fact suggeststhat the genes of the heme transport cluster were acquired byhorizontal gene transfer, simultaneously with the TonB genes. This may have represented a selective advantage, allowing efficient heme transport by the recipient strain . However, some differences in gene order are observed . Among the Vibrio species, V . anguillarumis unique in that the outer membrane receptor gene is linkedto the rest of the heme transport genes . Similarly, upstream of the huvX homologue, all other Vibrio species, as well as P . shigelloides, contain a gene which is transcribed in the same direction as huvX and huvZ homologues and is absent in the V . anguillarum heme uptake cluster . This gene codes for a putative coproporphyrinogen oxidase [it has been named HutW, PhuW, or HugW], an enzyme that converts coproporphyrinogen IIIinto protoporphyrin IX, one of the steps in the heme biosynthesispathway [31].

 
Finally, a gene [hupR] coding for a member of the LysR family of transcriptional activators, which regulates expression ofthe heme receptor, has been found in V . vulnificus [21] . Homologuesof this gene are encountered upstream of V . parahaemolyticushutA [22] and V . cholerae hutA [12] . However, the nucleotidesequence immediately upstream of V . anguillarum huvA does notcode for an HupR homologue [data not shown].

Phenotypic analysis of heme uptake system chromosomal mutants with hemin or hemoglobin as the only iron source. In order to evaluate the importance of individual genes of theheme uptake system in the utilization of hemin or hemoglobin,individual in-frame deletions of huvA, huvZ, huvX, huvB, huvC,and huvD and an in-frame deletion of the complete nine-genecluster were constructed . The H-775-3 strain, deficient in siderophoreanguibactin biosynthesis, was chosen in order to eliminate backgroundgrowth resulting from anguibactin-mediated iron uptake in iron-restricted media . Growth assays were carried out in triplicate, and results shown here are the means of three independent experiments . Strains were first assayed in an iron-sufficient medium with hemin [10µM] . No significant differences in growth rates were seenin iron-sufficient medium between mutant strains and V . anguillarum H-775-3 [Fig . 4A] . The same mutants were then grown in an iron-restrictedmedium with the iron chelator 2,2'-dipyridyl added at a concentrationof 100 µM and containing 10 µM hemin as the sole iron source . Under these conditions, no significant differences in growth were observed between the huvX strain and the parentalstrain, V . anguillarum H-775-3, with the slight differencesobserved likely due to a lower initial inoculum . This demonstratesthat this gene is not essential for the utilization of heminas the sole iron source in V . anguillarum . By contrast, therest of the assayed mutants were drastically affected in theirability to use hemin . Mutation of any huvAZBCD gene decreasedbacterial growth to minimal levels [Fig . 4B] . The huv strainshowed a reduction in growth with hemin as the sole iron sourcecomparable to that observed in the single huvA, -Z, -B, -C,and -D mutants [Fig. 4B] . The same results were obtained forall the assayed strains when hemoglobin was used as the ironsource instead of hemin [data not shown].

 
Utilization of heme compounds by E . coli EB53 and 101ESD complemented with V . anguillarum genes. The ability of V . anguillarum heme transport genes to allowthe use of hemin or hemoglobin as porphyrin and iron sourceswas evaluated in E . coli EB53 [aroB hemA] and in E . coli 101ESD [[entC-entA]], respectively . The aroB and entC-entA mutationsrender E . coli unable to produce its own siderophore, enterochelin, and the hemA mutation disrupts synthesis of heme . Therefore, E . coli EB53 cannot grow unless supplied with the heme biosynthetic precursor ALA . EB53 could satisfy the porphyrin deficiency by utilizing exogenously supplied hemin, as long as a genetic systemfor hemin uptake is provided . Similarly, 101ESD cannot growin the presence of iron chelators unless supplied with a utilizablesource of iron.

Growth around hemin [0.05 and 5 mM] or hemoglobin [1 mM] diskson nutrient agar plates [NB] was used to assay the use of porphyrin sources by E . coli EB53 . To test whether the introduction of the entire heme transport gene cluster would promote hemin and hemoglobin utilization as porphyrin sources, cosmid pML1, containing the entire heme transport region within a ca . 15-kb V . anguillarum H-775-3 genome fragment, was introduced into EB53 by triparental mating . This transformant utilized hemin and hemoglobin as porphyrin sources . A subclone of pML1 [pCAR121] containing only huvA proved to be sufficient to confer the utilization of hemin and hemoglobin as porphyrin sources upon E . coli EB53 . This indicates that E . coli EB53 [a K-12 derivative] encodes all the additional functions necessary for transporting and utilizing heme as a porphyrin when an outer membrane heme receptor is provided.None of other plasmid combinations assayed, in which huvA wasabsent, could complement EB53 [data not shown].

To determine which genes are necessary for the utilization of hemin and hemoglobin as iron sources, E . coli 101ESD was complemented with V . anguillarum genes and tested on CM9 minimal medium plates supplemented with 100 µM 2,2'-dipyridyl and with heminand hemoglobin supplied on paper disks . Results are summarizedin Table 4 . E . coli 101ESD transformed with plasmids containing huvA alone did not grow in this medium, indicating that other genes in addition to huvA are needed for the utilization of hemin or hemoglobin as an iron source . However, E . coli 101ESD transformed with cosmid pML1 utilized both compounds as iron sources . In order to determine the minimum genetic background necessary for utilization of heme as an iron source, differentgene combinations were assayed.

 
Heme iron utilization did not occur when strain 101ESD/pCAR121was transformed with pSML11 [huvZX] or with pCAR126 [huvX tonB exbBD huvB] . The complete nine-gene cluster was PCR amplified, cloned in pWKS30 to yield pSML23, and transformed into 101ESD.This strain utilized hemin and hemoglobin as iron sources, indicatingthat this capacity is encoded within the nine-gene cluster describedhere and that this utilization is not due to extra genes presentin pML1.

Complete tonB exbBD huvBCD genes cloned in pSML33 and transformed into strain 101ESD/pCAR121 allowed this strain to utilize hemin and hemoglobin as iron sources . However, 101ESD/pCAR121/pSML32and 101ESD/pCAR121/pSML34 failed to grow with hemin or hemoglobin.These results together demonstrate that tonB exbBD genes in combination with huvA are not sufficient for heme iron utilization unless huvBCD genes are also provided . It is feasible that huvBCDgenes are the crucial ones in combination with huvA to allowtransport of heme compounds into the cytoplasm of E . coli andfurther utilization as iron sources . The finding that tonB exbBDgenes need to be provided in the same plasmid together with downstream huvBCD genes in order to allow complementation may be explained by the existence of a single promoter upstreamof tonB, as has been reported for V . cholerae [29].

 
Utilization of hemin and heme-containing proteins as iron sourceshas been reported for V . anguillarum [23, 24], but the molecularmechanism supporting heme uptake is unknown . In a recent work,the V . anguillarum outer membrane heme receptor gene huvA wascharacterized, and a huvA mutant obtained by chemical mutagenesisshowed a reduction in virulence for fish [25] . In the presentstudy, we characterized a gene cluster in V . anguillarum thathas similarities with heme iron assimilation systems found inother Vibrio species and P . shigelloides, both at the aminoacid level and gene organization.

Homologues of V . anguillarum HuvZX proteins have been described, associated with the heme utilization systems of V . cholerae, V . vulnificus, Vibrio parahaemolyticus, and P . shigelloides,but to date their roles remain unascertained . In the presentstudy we report the mutational analysis of V . anguillarum huvZXgenes . Our results have shown that a huvX deletion mutant isable to use heme nearly as efficiently as the parental strain.This suggests that either huvX is not directly involved in theuse of heme as an iron source or additional V . anguillarum genesmay substitute for the function of huvX . However, deletion ofhuvZ drastically reduces the ability of the bacterium to growwith heme as the sole iron source, indicating that this geneis essential for heme iron utilization . A HuvZ-related activitymay be present in E . coli 101ESD, since the heme uptake systemof V . anguillarum can be easily reconstituted in 101ESD withoutHuvZ . However, the actual function of HuvZ remains unknown.A possibility would be that HuvZ is involved in removing ironfrom heme . It has been proposed that the oxidative cleavageof heme mediated by heme oxygenases is a mechanism for iron acquisition for some bacteria . Heme oxygenase genes have been described recently for Neisseria meningitidis [44], and Corynebacteriumdiphtheriae [42], but no significant homology exists betweenthese described heme oxygenase genes and HuvZ . Recently, Wyckoffet al . [43] demonstrated that hutZ [homologous to huvZ] is also essential for heme iron utilization in V . cholerae . These authors could not demonstrate a heme oxygenase activity for HutZ, suggesting that it may act as a heme carrier or storage protein . Further studies are needed in order to ascertain the role played byHuvZ in the utilization of heme as an iron source.

V . anguillarum huvBCD genes are essential for heme uptake . Nonpolar deletions in any of these three genes drastically reduce the growth of V . anguillarum with hemin or hemoglobin as the sole iron source . In contrast, it has been reported that V . cholerae hutB, hutC, or hutD mutants still retain significant growth when hemin is used as the only iron source [29] . Thus, it ispossible that proteins of other transport systems of V . choleraecan substitute for the physiological role of either HutB, -C,or -D, while in V . anguillarum each one of these proteins isessential for heme uptake.

Complementation studies carried out with E . coli EB53 showed that the V . anguillarum outer membrane heme receptor HuvA is sufficient for utilization of heme compounds as porphyrin sources. Complementation of E . coli heme-deficient mutants with an outer membrane heme receptor provided in trans has been previously reported [36] . Interestingly, the P . shigelloides heme receptorgene hugA could not complement an E . coli DHE-1 hemA mutantfor the use of heme as a porphyrin source unless P . shigelloidestonB-exbB-exbD genes were also provided in trans [14].

In the use of heme compounds as an iron source, huvA plus tonB exbBD huvBCD are needed to reconstitute the V . anguillarum heme transport system in E . coli 101ESD [an HB101 derivative] . Similarly,Occhino et al . [29] reported that utilization of hemin as aniron source can be reconstituted in E . coli 1017 [HB101 derivative,ent] with V . cholerae hutA, tonB exbBD, and hutBCD genes . Incontrast, Henderson et al . [14] observed that a plasmid containing hugWXZ was necessary in addition to hugA and tonB-exbBD to reconstitutethe P . shigelloides heme iron utilization system in E . coli1017.

We hypothesize that the V . anguillarum huvBCD genes are crucial for heme iron utilization in E . coli 101ESD but the tonB and exbBD genes are not . Complementation of EB53 for heme utilization as a porphyrin source did not depend on the presence of the V . anguillarum TonB system in trans, which means that huvA aloneis active in an E . coli background . We propose that hutBCD genesare transcribed from a promoter located upstream of the tonBgene, and thus actual complementation for heme iron utilizationis achieved only when the six genes tonB, exbBD, and huvBCD,are provided together on a plasmid . It remains to be explainedwhy heme utilization as a porphyrin source in E . coli EB53 doesnot depend on huvBCD genes while utilization as an iron sourcein E . coli 101ESD does require huvBCD . This could be due toa difference in strain background, as for example, the presenceof an inner membrane transporter in EB53.

As reported by Stojiljkovic and Hantke [36], the Y . enterocoliticaouter membrane heme receptor HemR alone is sufficient to complementE . coli for utilization of heme as a porphyrin source, and itis possible that hemin, once in the periplasm, can be incorporatedinto cytochromes located in the cytoplasmic membrane [10] . However,utilization of heme compounds as iron sources would be feasibleonly as long as the heme compound is transported from the periplasmicspace into the cytoplasm, where it is expected that additionalproteins are implicated in degradation of the heme moleculeand in the release of iron . As proposed by Stojiljkovic andHantke [36], the difference between porphyrin and iron utilizationfrom heme may be merely quantitative, being the amount of hemeneeded to satisfy the cell's requirements for iron, much largerthan the amount necessary as a porphyrin source . This beingthe case, E . coli EB53 complemented with V . anguillarum HuvAcould transport trace amounts of heme as a porphyrin sourceinto the cytoplasm via nonspecific E . coli ABC transporters.However, heme iron utilization could be effective only as longas a specific ABC transporter [HuvBCD] is provided.

The gene coding for the V . anguillarum outer membrane heme receptor is linked to the rest of heme transport genes . Such a spatial organization is unusual in other species of the family Vibrionaceae, where the outer membrane receptor gene is separated from the rest of the transport genes by hundreds of kilobases [1, 12,22] . Other differences in the V . anguillarum heme uptake clusterinclude the absence of a putative coproporphyrinogen oxidasegene and a gene for a LysR transcriptional activator homologue,which are present in V . vulnificus, V . cholerae, and V . parahaemolyticus[1, 12, 22] . It is possible that the heme transport clusteroriginally included homologues of these genes, which eventuallyunderwent a spatial reorganization in the genome of V . anguillarum.

In conclusion, we have shown in this study that the heme uptake cluster of V . anguillarum H-775-3 includes nine genes, fiveof which proved to be essential for utilization of heme as aniron source . The gene arrangement of the V . anguillarum hemeuptake cluster has unique features which differentiate it fromhomologous clusters found in other gram-negative bacteria.

 
We gratefully acknowledge J . H . Crosa, K . E . Klose, and V . Braunfor providing strains and plasmids.

This work was supported by grants AGL2000-0492 and AGL-2003-00086 from the Ministry of Science and Technology of Spain [cofundedby the FEDER Programme from the European Union] and grant PGIDT01PXI26202PN from Xunta de Galicia to M.L.L.

 
* Corresponding author . Mailing address: Department of Microbiology and Parasitology, Institute of Aquaculture and Faculty of Biology, University of Santiago de Compostela, Santiago de Compostela 15782, Spain . Phone: 34-981563100, ext . 16080 . Fax: 34-981547165 . E-mail: mlemos@usc.es .

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