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The ability to utilize the iron bound by high-affinity iron-binding proteins in the vertebrate host is an important virulence factor for the marine fish pathogen Vibrio anguillarum. Virulence in septicemic infections is due to the presence of a highly efficient plasmid-encoded iron transport system. AngR, a 110-kDa protein component of this system, appears to play a role in both regulation of the expression of the iron transport genes fatDCBA and the production of the siderophore anguibactin. Therefore, study of the expression of the angR gene and the properties of its product, the AngR protein, may contribute to the understanding of the mechanisms of virulence of this pathogen. In this work, we present genetic and molecular evidence from transposition mutagenesis experiments and RNA analysis that angR, which maps immediately downstream of the fatA gene, is part of a polycistronic transcript that also includes the iron transport genes fatDCBA and angT, a gene located downstream of angR which showed domain homology to certain thioesterases involved in nonribosomal peptide synthesis of siderophores and antibiotics. In order to dissect the specific domains of AngR associated with regulation of iron transport gene expression, anguibactin production, and virulence, we also generated a panel of site-directed angR mutants, as well as deletion derivatives. Both virulence and anguibactin production were dramatically affected by each one of the angR modifications. In contrast to the need for an intact AngR molecule for anguibactin production and virulence, the regulation of iron transport gene expression does not require the entire AngR molecule, since truncation of the carboxy terminus carrying the nonribosomal peptide synthetase cores, as well as the site-directed mutations, resulted in derivatives that retained their ability to regulate gene expression which was only abolished after truncation of amino-terminal sequences containing helix-turn-helix and leucine zipper motifs and a specialized heterocyclization and condensation domain found in certain nonribosomal peptide synthetases. The evidence, while not rigorously eliminating the possibility that a separate regulatory polypeptide exists and is encoded somewhere within the 5'-end region of the angR gene, strongly supports the idea that AngR is a bifunctional protein and that it plays an essential role in the virulence mechanisms of V. anguillarum. We also show in this study that the angT gene, found downstream of angR, intervenes in the mechanism of anguibactin production but is not essential for virulence or iron transport gene expression.

Vibrio anguillarum virulence is associated with the presence of a plasmid-mediated iron-uptake system expressed under iron-limiting conditions, which consists of the siderophore anguibactin and specific iron-transport proteins. This system is maximally expressed under iron-limiting conditions and requires the AngR protein that acts as a positive regulator of anguibactin biosynthesis and also possess an EntE-like enzymatic function that may play a role in anguibactin biosynthesis. In this work, we demonstrate that in addition to possessing these functions related to anguibactin production, AngR also positively regulated transcription of the iron-transport genes fatA and fatB. We also show that transcription of angR is repressed by Fur under iron-rich conditions. In addition, we present evidence that anguibactin itself enhanced transcription of the iron-transport genes fatA and fatB, independently of AngR and the trans-acting factor (TAF) product(s). The presence of either AngR (together with the TAF product(s)) or anguibactin alone led to a partial level of expression of the iron-transport genes fatA and fatB, while full expression is achieved when AngR, the TAF products and anguibactin are all present.

The pJM1-encoded genes fatDCBA are essential for iron acquisition via the siderophore anguibactin. Sequence analysis indicated that the open reading frame corresponding to the fatB gene possesses domains that are characteristic of periplasmic proteins that bind the ferric siderophore. In this work, a monospecific antiserum against an oligopeptide containing the last 27 amino acids of the carboxy-terminal region from this open reading frame was used to demonstrate that fatB encodes a 35 kDa protein that is essential for iron transport. By using this antibody we were able to demonstrate that expression of the fatB gene is negatively regulated by the Fur protein at high iron concentrations. Conversely, its expression was positively regulated by the combined action of the AngR protein and products of the TAF region. FatB, the product of the fatB gene, is isolated with the membrane fraction. In accordance with these findings is the fact that the first 23 amino acid residues of this protein have the properties of a lipoprotein signal sequence. The lipoprotein nature of FatB is supported by the fact that treatment of Vibrio anguillarum cells with globomycin, an inhibitor of the lipoprotein signal peptidase, results in the accumulation of a 38 kDa proFatB precursor protein.

We have identified and sequenced an hdc gene in the Vibrio anguillarum plasmid pJM1 which encodes a histidine decarboxylase enzyme and is an essential component for the biosynthesis of anguibactin. The open reading frame corresponds to a protein of 386 amino acids with a calculated molecular mass of 44,259.69 Da. The amino acid sequence has extensive homology with the pyridoxal-P-dependent histidine decarboxylases of Morganella morganii, Klebsiella planticola, and Enterobacter aerogenes. Tn3-HoHo1 transposition mutagenesis of the hdc gene present in a recombinant clone carrying the entire pJM1 iron uptake region produced two derivatives, one with the lacZ gene in the same orientation as the direction of hdc transcription and the other with the lacZ gene in the opposite orientation. A. V. anguillarum strain harbouring one of the mutated derivatives was unable to grow under iron-limiting conditions and did not produce anguibactin. Therefore, the hdc gene must play a role in the biosynthetic pathway of this siderophore and consequently in conferring the high virulence phenotype to this bacterium. The role of histidine decarboxylase in biosynthesis of anguibactin was confirmed by the fact that growth under iron starvation was restored by addition of histamine to the medium. The presence of anguibactin was also demonstrated in supernatants from cultures of the hdc mutant strains grown under iron starvation with the addition of histamine, further confirming that histamine is a precursor in the biosynthesis of the siderophore.(ABSTRACT TRUNCATED AT 250 WORDS)

We have isolated a recombinant clone harboring the chromosomal aroC gene, encoding chorismate synthase, from Vibrio anguillarum 775 by complementation of the Escherichia coli aroC mutant AB2849 which was transfected with a cosmid gene bank of the plasmidless V. anguillarum H775-3. The nucleotide sequence was determined, and an open reading frame that corresponds to a protein of 372 amino acids was found. The calculated mass of 40,417 Da was correlated with the size of the V. anguillarum aroC product detected in vitro. The homology of the V. anguillarum aroC gene to the aroC genes of E. coli and Salmonella typhi is 68% at the nucleotide level and 78% at the protein level. The expression of the aroC transcript is not regulated by iron, as determined by Northern (RNA) blot hybridization analysis. After insertion of an antibiotic resistance gene cassette within the cloned aroC gene, an aroC mutant of V. anguillarum was generated by allelic exchange. This mutant is deficient in the production of 2,3-dihydroxybenzoic acid (2,3-DHBA). Our bioassay and complementation experiments with this mutant demonstrate that the chromosome-mediated 2,3-DHBA is a precursor of the pJM1 plasmid-mediated siderophore anguibactin.

The siderophore anguibactin is produced in vivo in a diffusible form and is an important factor in the virulence of Vibrio anguillarum. The natural isolate V. anguillarum 531A is a hyperproducer of anguibactin when compared with the prototype strain V. anguillarum 775. The angR gene was found to be responsible for this difference in levels of anguibactin produced. Nucleotide sequence analysis showed that the angR531A differed in a single nucleotide from the angR775 present in the prototype plasmid pJM1. This nucleotide substitution resulted in a change in amino acid 267 from His in strain 775 to Asn in strain 531A. This amino acid is located in a region between one of the two helix-turn-helix domains and the neighboring leucine zipper. Mutations to replace His with either Leu or Gln, generated by site-directed mutagenesis, in amino acid 267 resulted in strains for which the MIC of the iron chelator ethylenediamine di(o-hydroxyphenyl) acetic acid were lower than for the proptotype 775 but higher than for iron uptake-deficient strains. In addition to its transcriptional activating function, AngR also complemented a mutation in the Escherichia coli entE gene, which encodes the enterobactin biosynthetic enzyme 2,3-dihydroxybenzoate-AMP ligase. Therefore, AngR may also function in V. anguillarum as an EntE-like enzyme for the biosynthesis of anguibactin.

Anguibactin, a siderophore produced by cells of Vibrio anguillarum 775 harboring the pJM1 plasmid, has now been isolated from the supernatants of iron-deficient cultures. This iron-reactive material was purified by adsorption onto an XAD-7 resin and subsequent gel filtration on a Sephadex LH-20 column. The resulting neutral compound produced an ion at m/z 348 in mass spectrometry and contained one sulfur, four oxygen, and four nitrogen atoms as determined by elemental analysis. Its strong UV absorbance and blue fluorescence were suggestive of a phenolic moiety. In colorimetric reactions anguibactin behaved like a catechol. The catechol assignment was supported by the appearance of a new absorption band at 510 nm in the ferric complex and by the appearance of peaks at 1,367, 1,447, 1,469, and 1,538 cm-1 in the resonance Raman spectrum. In addition, the infrared spectrum gave evidence of a secondary amide function, but no free carboxylic acid or hydroxamic acid groups were observed. A third iron-ligating group was suggested by the liberation of three protons during iron binding; mass spectrometry of the resulting material yielded a molecular ion characteristic of a 1:1 complex of ferric anguibactin. The purified anguibactin exhibited specific growth-promoting activity under iron-limiting conditions for a siderophore-deficient mutant of V. anguillarum 775(pJM1). A novel structure for anguibactin was indicated by the failure of a large number of known siderophores and synthetic chelators to yield a similar type of specific cross-feeding in the V. anguillarum bioassay.

The possession of specialized iron transport systems may be crucial for bacteria to override the iron limitation imposed by the host or the environment. One of the most commonly found strategies evolved by microorganisms is the production of siderophores, low-molecular-weight iron chelators that have very high constants of association for their complexes with iron. Thus, siderophores act as extracellular solubilizing agents for iron from minerals or organic compounds, such as transferrin and lactoferrin in the host vertebrate, under conditions of iron limitation. Transport of iron into the cell cytosol is mediated by specific membrane receptor and transport systems which recognize the iron-siderophore complexes. In this review I have analyzed in detail three siderophore-mediated iron uptake systems: the plasmid-encoded anguibactin system of Vibrio anguillarum, the aerobactin-mediated iron assimilation system present in the pColV-K30 plasmid and in the chromosomes of many enteric bacteria, and the chromosomally encoded enterobactin iron uptake system, found in Escherichia coli, Shigella spp., Salmonella spp., and other members of the family Enterobacteriaceae. The siderophore systems encoded by Pseudomonas aeruginosa, namely, pyochelin and pyoverdin, as well as the siderophore amonabactin, specified by Aeromonas hydrophila, are also discussed. The potential role of siderophore-mediated systems as virulence determinants in the specific host-bacteria interaction leading to disease is also analyzed with respect to the influence of these systems in the expression of other factors, such as toxins, in the bacterial virulence repertoire.

The iron-transport genes from the pJM1 plasmid of Vibrio anguillarum have been cloned and sequenced. Five open-reading frames have been identified, one of which encodes the outer membrane receptor for ferric anguibactin, OM2. This coding region corresponds to a protein of 726 amino acids with a Mr of 78,777. The protein has a hydrophobic signal sequence of 35 amino acids and a potential membrane-associated hydrophobic region at the carboxyl terminus. A 2.3-kilobase iron-regulated mRNA was transcribed from this region in vivo. The four other open-reading frames were shown to be involved in the regulation of OM2 expression and in iron transport by the use of insertion mutagenesis and complementation analysis. One of these open-reading frames, ORF3, encodes a 40-kDa polypeptide which, as deduced from the amino acid sequence and the hydropathy plot, is likely to be membrane-associated and together with OM2 may play a role in the transport of iron into the cell cytosol.

We have identified a 110-kDa polypeptide that has a trans-acting regulatory activity on the expression of the pJM1 plasmid iron-uptake genes in Vibrio anguillarum. This protein is encoded by the angR gene and maps in a 3.6-kilobase-pair pJM1 DNA region located downstream of the iron transport genes. Full expression of this gene occurs under iron-limiting conditions and requires a 2.9-kilobase-pair upstream region in cis that maps within the coding region of the OM2 outer membrane protein, essential for the transport of iron into the cell cytosol. Determination of the siderophore anguibactin levels as well as analysis of specific transcripts for anguibactin biosynthetic genes demonstrated that AngR and another transcriptional activator, Taf, regulate in a synergistic fashion the level of anguibactin production by activation of transcription of the anguibactin biosynthetic genes under iron-limiting conditions.

We describe in this work a new iron uptake system encoded by chromosomal genes in pathogenic strains of Vibrio anguillarum. This iron uptake system differs from the plasmid-encoded anguibactin-mediated system present in certain strains of V. anguillarum in several properties. The siderophore anguibactin is not utilized as an external siderophore, and although characteristic outer membrane proteins are synthesized under iron-limiting conditions, these are not related to the plasmid-mediated outer membrane protein OM2 associated with ferric anguibactin transport. Furthermore, the siderophore produced by the plasmidless strains may be functionally related to enterobactin as demonstrated by bioassays with enterobactin-deficient mutants, although its behavior under various chemical treatments suggested major differences from that siderophore. Hybridization experiments suggested that the V. anguillarum chromosome-mediated iron uptake system is unrelated genetically to either the anguibactin or enterobactin-associated iron assimilation systems.

The virulence of the fish pathogen Vibrio anguillarum 775 is mediated by the pJM1 plasmid-specified iron uptake system which is expressed under conditions of iron limitation. Other V. anguillarum strains isolated from various geographical locations harbor plasmids that are highly related to pJM1 and that are also associated with the high-virulence phenotype of these strains. In this work, we found that a pJM1-like plasmid, pJHC1, from one of these virulent strains encoded an iron uptake system that resulted in an increased level of production of the siderophore anguibactin. The gene(s) responsible for increased anguibactin production was included within the iron uptake region of plasmid pJHC1. The cloned iron uptake regions of pJHC1 and pJM1 possessed identical restriction endonuclease maps, suggesting that the DNA region encoding those genes in pJHC1 may have diverged subtly from that in pJM1. Analysis of the iron uptake system from other V. anguillarum strains carrying pJM1-like plasmids demonstrated that strains originating from diseased fish from the Atlantic coast carry plasmids encoding an increased-siderophore-production phenotype, while strains isolated from Pacific Ocean locations behaved as the 775 strain.

Clones carrying the iron uptake region of the Vibrio anguillarum plasmid pJM1 were subjected to insertion mutagenesis, using transposon Tn3::HoHo1 which carries a promoterless lacZ gene and can thus generate lacZ transcriptional fusions if inserted downstream from an indigenous promoter. Four classes of insertion mutants were obtained based on the level of expression of components of the iron uptake system, and six genetic units were defined according to the phenotype of the mutants. Five of the six genetic units were crucial for biosynthesis of the siderophore anguibactin. Insertions in the remaining genetic unit led to an iron uptake-deficient phenotype and showed either reduced levels of the outer membrane protein OM2 as well as anguibactin activity or a complet shutoff of both OM2 and anguibactin biosyntheses. Analysis of beta-galactosidase production by cells carrying the lacZ fusion derivatives identified iron-regulated and constitutive transcriptional units as well as their orientation in the genetic units. Molecular cloning of pJM1 plasmid DNA noncontiguous to the iron uptake region also identified genetic determinants for a trans-acting factor required for full expression of anguibactin activity. Evidence obtained from bioassays, spectrophotometric measurements, and the lacZ fusion mutants suggested that the trans-acting factor is a novel activator of siderophore biosynthesis at the transcriptional level.