Identification and characterization of a periplasmic trilactone esterase,Cee, revealed unique features of ferric enterobactin acquisition in Campylobacter

Ferric enterobactin (FeEnt) acquisition is a highly efficient and conserved iron scavenging system in Gram‐negative bacteria. Recently, we have characterized two FeEnt receptors (CfrA and CfrB) in Campylobacter jejuni and C. coli, the enteric human pathogens that do not produce any siderophores. In this study, whole‐genome sequencing and comparative genomic analysis identified a unique Ent trilactone esterase Cee (Cj1376) in C. jejuni. Genomic analysis and biochemical assay strongly suggested that Cee is the sole trilactone esterase in C. jejuni. Thin‐layer chromatography and HPLC analyses showed high efficiency of the purified Cee to hydrolyse Ent. Three Cee homologues previously characterized from other bacteria (IroE, IroD and Fes) were also purified and analysed together with Cee, indicating that Cee, Fes and IroD displayed similar hydrolysis dynamics for both apo and ferric forms of Ent while IroE catalysed Ent inefficiently. Unlike cytoplasmic Fes and IroD, Cee is localized in the periplasm as demonstrated by immunoblotting using Cee‐specific antibodies. Genetic manipulation of diverse Campylobacter strains demonstrated that Cee is not only essential for CfrB‐dependent FeEnt acquisition but also involved in CfrA‐dependent pathway. Together, this study identified and characterized a novel periplasmic trilactone esterase and suggested a new model of FeEnt acquisition in Campylobacter.     

Chlorophyllase as a serine hydrolase: identification of a putative catalytic triad

Chlorophyllases (Chlases), cloned so far, contain a lipase motif with the active serine residue of the catalytic triad of triglyceride lipases. Inhibitors specific for the catalytic serine residue in serine hydrolases, which include lipases effectively inhibited the activity of the recombinant Chenopodium album Chlase (CaCLH). From this evidence we assumed that the catalytic mechanism of hydrolysis by Chlase might be similar to those of serine hydrolases that have a catalytic triad composed of serine, histidine and aspartic acid in their active site. Thus, we introduced mutations into the putative catalytic residue (Ser162) and conserved amino acid residues (histidine, aspartic acid and cysteine) to generate recombinant CaCLH mutants. The three amino acid residues (Ser162, Asp191 and His262) essential for Chlase activity were identified. These results indicate that Chlase is a serine hydrolase and, by analogy with a plausible catalytic mechanism of serine hydrolases, we proposed a mechanism for hydrolysis catalyzed by Chlase.     

Ligand-induced structural changes in adenosine 5'-phosphosulfate kinase from Penicillium chrysogenum

Adenosine 5'-phosphosulfate (APS) kinase catalyzes the second reaction in the two-step, ATP-dependent conversion of inorganic sulfate to 3'-phosphoadenosine 5'-phosphosulfate (PAPS). PAPS serves as the sulfuryl donor for the biosynthesis of all sulfate esters and also as a precursor of reduced sulfur biomolecules in many organisms. Previously, we determined the crystal structure of ligand-free APS kinase from the filamentous fungus, Penicillium chrysogenum [MacRae et al. (2000) Biochemistry 39, 1613-1621]. That structure contained a protease-susceptible disordered region ("mobile lid"; residues 145-170). Addition of MgADP and APS, which together promote the formation of a nonproductive "dead-end" ternary complex, protected the lid from trypsin. This report presents the 1.43 A resolution crystal structure of APS kinase with both ADP and APS bound at the active site and the 2.0 A resolution structure of the enzyme with ADP alone bound. The mobile lid is ordered in both complexes and is shown to provide part of the binding site for APS. That site is formed primarily by the highly conserved Arg 66, Arg 80, and Phe 75 from the protein core and Phe 165 from the mobile lid. The two Phe residues straddle the adenine ring of bound APS. Arg 148, a completely conserved residue, is the only residue in the mobile lid that interacts directly with bound ADP. Ser 34, located in the apex of the P-loop, hydrogen-bonds to the 3'-OH of APS, the phosphoryl transfer target. The structure of the binary E.ADP complex revealed further changes in the active site and N-terminal helix that occur upon the binding/release of (P)APS.     

The PE-PPE domain in mycobacterium reveals a serine α/β hydrolase fold and function: an in-silico analysis

The PE and PPE proteins first reported in the genome sequence of Mycobacterium tuberculosis strain H37Rv are now identified in all mycobacterial species. The PE-PPE domain (Pfam ID: PF08237) is a 225 amino acid residue conserved region located towards the C-terminus of some PE and PPE proteins and hypothetical proteins. Our in-silico sequence analysis revealed that this domain is present in all Mycobacteria, some Rhodococcus and Nocardia farcinica genomes. This domain comprises a pentapeptide sequence motif GxSxG/S at the N-terminus and conserved amino acid residues Ser, Asp and His that constitute a catalytic triad characteristic of lipase, esterase and cutinase activity. The fold prediction and comparative modeling of the 3-D structure of the PE-PPE domain revealed a "serine α/β hydrolase" structure with a central β-sheet flanked by α-helices on either side. The structure comprises a lid insertion with a closed structure conformation and has a solvent inaccessible active site. The oxyanion hole that stabilizes the negative charge on the tetrahedral intermediate has been identified. Our findings add to the growing list of serine hydrolases in mycobacterium, which are essential for the maintenance of their impermeable cell wall and virulence. These results provide the directions for the design of experiments to establish the function of PE and PPE proteins.     

The structure of the ferric siderophore binding protein FhuD complexed with gallichrome

Siderophore binding proteins play a key role in the uptake of iron in many gram-positive and gram-negative bacteria. FhuD is a soluble periplasmic binding protein that transports ferrichrome and other hydroxamate siderophores. The crystal structure of FhuD from Escherichia coli in complex with the ferrichrome homolog gallichrome has been determined at 1.9 ? resolution, the first structure of a periplasmic binding protein involved in the uptake of siderophores. Gallichrome is held in a shallow pocket lined with aromatic groups; Arg and Tyr side chains interact directly with the hydroxamate moieties of the siderophore. FhuD possesses a novel fold, suggesting that its mechanisms of ligand binding and release are different from other structurally characterized periplasmic ligand binding proteins.     

Tuning the substrate selectivity of meta-cleavage product hydrolase by domain swapping

meta-Cleavage product (MCP) hydrolases can catalyze relatively low reactive carbon–carbon bond hydrolysis of products, which are derived from the meta-cleavage of catechols. The strict substrate selectivity of MCP hydrolases attracts an interest to understand the determinants of substrate specificity. Compared with conventional site-directed mutagenesis, domain swapping is an effective strategy to explore substrate specificity due to the large-scale reorganization of three-dimensional structure. In the present study, the hybrid MCP hydrolases BphD_LidA and MfphA_LidD were constructed by exchanging the lid domain of two parental enzymes MfphA and BphD. The residues Gly130/Ala196 (MfphA) and Gly136/Ala211 (BphD) were selected as crossover points according to structural disruption score analysis and molecular dynamics simulations. It was shown that the hybrid enzymes exhibited similar substrate selectivity with the parent enzyme providing the lid domain. Docking studies suggested that the lid domain may play a key role in determining substrate specificity by reshaping the active pocket and modulating the orientation of the substrate.     

Roles of active site residues in Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase

In Pseudomonas aeruginosa, the dual-specificity enzyme phosphomannomutase/phosphoglucomutase catalyzes the transfer of a phosphoryl group from serine 108 to the hydroxyl group at the 1-position of the substrate, either mannose 6-P or glucose 6-P. The enzyme must then catalyze transfer of the phosphoryl group on the 6-position of the substrate back to the enzyme. Each phosphoryl transfer is expected to require general acid-base catalysis, provided by amino acid residues at the enzyme active site. An extensive survey of the active site residues by site-directed mutagenesis failed to identify a single key residue that mediates the proton transfers. Mutagenesis of active site residues Arg20, Lys118, Arg247, His308, and His329 to residues that do not contain ionizable groups produced proteins for which V(max) was reduced to 4-12% of that of the wild type. The fact that no single residue decreased catalytic activity more significantly, and that several residues had similar effects on V(max), suggested that the ensemble of active site amino acids act by creating positive electrostatic potential, which serves to depress the pK of the substrate hydroxyl group so that it binds in ionized form at the active site. In this way, the necessity of positioning the reactive hydroxyl group near a specific amino acid residue is avoided, which may explain how the enzyme is able to promote catalysis of both phosphoryl transfers, even though the 1- and 6-positions do not occupy precisely the same position when the substrate binds in the two different orientations in the active site. When Ser108 is mutated, the enzyme retains a surprising amount of activity, which has led to the suggestion that an alternative residue becomes phosphorylated in the absence of Ser108. (31)P NMR spectra of the S108A protein confirm that it is phosphorylated. Although the S108A/H329N protein had no detectable catalytic activity, the (31)P NMR spectra were not consistent with a phosphohistidine residue.     

The "rhodanese" fold and catalytic mechanism of 3-mercaptopyruvate sulfurtransferases: crystal structure of SseA from Escherichia coli

3-Mercaptopyruvate sulfurtransferases (MSTs) catalyze, in vitro, the transfer of a sulfur atom from substrate to cyanide, yielding pyruvate and thiocyanate as products. They display clear structural homology with the protein fold observed in the rhodanese sulfurtransferase family, composed of two structurally related domains. The role of MSTs in vivo, as well as their detailed molecular mechanisms of action have been little investigated. Here, we report the crystal structure of SseA, a MST from Escherichia coli, which is the first MST three-dimensional structure disclosed to date. SseA displays specific structural differences relative to eukaryotic and prokaryotic rhodaneses. In particular, conformational variation of the rhodanese active site loop, hosting the family invariant catalytic Cys residue, may support a new sulfur transfer mechanism involving Cys237 as the nucleophilic species and His66, Arg102 and Asp262 as residues assisting catalysis.     

Structure of Aspergillus niger epoxide hydrolase at 1.8 A resolution: implications for the structure and function of the mammalian microsomal class of epoxide hydrolases

Background: Epoxide hydrolases have important roles in the defense of cells against potentially harmful epoxides. Conversion of epoxides into less toxic and more easily excreted diols is a universally successful strategy. A number of microorganisms employ the same chemistry to process epoxides for use as carbon sources. Results: The X-ray structure of the epoxide hydrolase from Aspergillus niger was determined at 3.5 A resolution using the multiwavelength anomalous dispersion (MAD) method, and then refined at 1.8 A resolution. There is a dimer consisting of two 44 kDa subunits in the asymmetric unit. Each subunit consists of an alpha/beta hydrolase fold, and a primarily helical lid over the active site. The dimer interface includes lid-lid interactions as well as contributions from an N-terminal meander. The active site contains a classical catalytic triad, and two tyrosines and a glutamic acid residue that are likely to assist in catalysis. Conclusions: The Aspergillus enzyme provides the first structure of an epoxide hydrolase with strong relationships to the most important enzyme of human epoxide metabolism, the microsomal epoxide hydrolase. Differences in active-site residues, especially in components that assist in epoxide ring opening and hydrolysis of the enzyme-substrate intermediate, might explain why the fungal enzyme attains the greater speeds necessary for an effective metabolic enzyme. The N-terminal domain that is characteristic of microsomal epoxide hydrolases corresponds to a meander that is critical for dimer formation in the Aspergillus enzyme.     

Identification of critical residues of the mycobacterial dephosphocoenzyme a kinase by site-directed mutagenesis

Dephosphocoenzyme A kinase performs the transfer of the γ-phosphate of ATP to dephosphocoenzyme A, catalyzing the last step of coenzyme A biosynthesis. This enzyme belongs to the P-loop-containing NTP hydrolase superfamily, all members of which posses a three domain topology consisting of a CoA domain that binds the acceptor substrate, the nucleotide binding domain and the lid domain. Differences in the enzymatic organization and regulation between the human and mycobacterial counterparts, have pointed out the tubercular CoaE as a high confidence drug target (HAMAP database). Unfortunately the absence of a three-dimensional crystal structure of the enzyme, either alone or complexed with either of its substrates/regulators, leaves both the reaction mechanism unidentified and the chief players involved in substrate binding, stabilization and catalysis unknown. Based on homology modeling and sequence analysis, we chose residues in the three functional domains of the enzyme to assess their contributions to ligand binding and catalysis using site-directed mutagenesis. Systematically mutating the residues from the P-loop and the nucleotide-binding site identified Lys14 and Arg140 in ATP binding and the stabilization of the phosphoryl intermediate during the phosphotransfer reaction. Mutagenesis of Asp32 and Arg140 showed catalytic efficiencies less than 5-10% of the wild type, indicating the pivotal roles played by these residues in catalysis. Non-conservative substitution of the Leu114 residue identifies this leucine as the critical residue from the hydrophobic cleft involved in leading substrate, DCoA binding. We show that the mycobacterial enzyme requires the Mg(2+) for its catalytic activity. The binding energetics of the interactions of the mutant enzymes with the substrates were characterized in terms of their enthalpic and entropic contributions by ITC, providing a complete picture of the effects of the mutations on activity. The properties of mutants defective in substrate recognition were consistent with the ordered sequential mechanism of substrate addition for CoaE.     

Amino acid variability in the peptide composition of a suite of amphiphilic peptide siderophores from an open ocean Vibrio species

In response to iron-depleted aerobic conditions, bacteria often secrete low molecular weight, high-affinity iron(III)-complexing ligands, siderophores, to solubilize and sequester iron(III). Many marine siderophores are amphiphilic and are produced in suites, wherein each member within a particular suite has the same iron(III)-binding polar head group which is appended by one or two fatty acids of differing length, degree of unsaturation, and degree of hydroxylation, establishing the suite composition. We report the isolation and structural characterization of a suite of siderophores from marine bacterial isolate Vibrio sp. Nt1. On the basis of structural analysis, this suite of siderophores, the moanachelins, is amphiphilic and composed of two N-acetyl-N-hydroxy-d-ornithines, one N-acetyl-N-hydroxy-l-ornithine, and either a glycine or an l-alanine, appended with various saturated and unsaturated fatty acid tails. The variation in the small side-chain amino acid is the first occurrence of variation in the peptidic head group structure of a set of siderophores produced by a single bacterium.     

Regulation of c-Fes tyrosine kinase activity by coiled-coil and SH2 domains: analysis with Saccharomyces cerevisiae

The c-Fes protein-tyrosine kinase regulates the growth and differentiation of diverse cell types, including myeloid hematopoietic cells, vascular endothelial cells, and neurons. Structurally, Fes is composed of a unique N-terminal region with coiled-coil oligomerization motifs, followed by SH2 and kinase domains. Although Fes kinase activity is tightly regulated in cells, the structural basis for its negative regulation is not clear. In this report, c-Fes was expressed in Saccharomyces cerevisiae to determine whether regulation is kinase-intrinsic or dependent upon protein factors found in mammalian cells. Wild-type Fes kinase activity was completely repressed in yeast and did not affect cell growth. Mutation or deletion of the more N-terminal c-Fes coiled-coil domain reversed negative regulation, leading to strong kinase activation and suppression of yeast cell growth. Similarly, replacement of the wild-type SH2 domain with that of v-Src induced strong kinase activation and the growth-inhibitory phenotype. Immunoblotting with phosphospecific antibodies shows that activation of Fes by either mechanism induced autophosphorylation of the activation loop tyrosine residue (Tyr 713). These data support the idea that Fes naturally adopts an inactive conformation in vivo, and that maintenance of the inactive structure requires the coiled-coil and SH2 domains.     

Anaerobic sulfatase-maturating enzymes, first dual substrate radical S-adenosylmethionine enzymes

Sulfatases are a major group of enzymes involved in many critical physiological processes as reflected by their broad distribution in all three domains of life. This class of hydrolases is unique in requiring an essential post-translational modification of a critical active-site cysteine or serine residue to C(alpha)-formylglycine. This modification is catalyzed by at least three nonhomologous enzymatic systems in bacteria. Each enzymatic system is currently considered to be dedicated to the modification of either cysteine or serine residues encoded in the sulfatase-active site and has been accordingly categorized as Cys-type and Ser-type sulfatase-maturating enzymes. We report here the first detailed characterization of two bacterial anaerobic sulfatase-maturating enzymes (anSMEs) that are physiologically responsible for either Cys-type or Ser-type sulfatase maturation. The activity of both enzymes was investigated in vivo and in vitro using synthetic substrates and the successful purification of both enzymes facilitated the first biochemical and spectroscopic characterization of this class of enzyme. We demonstrate that reconstituted anSMEs are radical S-adenosyl-l-methionine enzymes containing a redox active [4Fe-4S](2+,+) cluster that initiates the radical reaction by binding and reductively cleaving S-adenosyl-l-methionine to yield 5 '-deoxyadenosine and methionine. Surprisingly, our results show that anSMEs are dual substrate enzymes able to oxidize both cysteine and serine residues to C(alpha)-formylglycine. Taken together, the results support a radical modification mechanism that is initiated by hydrogen abstraction from a serine or cysteine residue located in an appropriate target sequence.     

Unusual catalytic triad of Escherichia coli outer membrane phospholipase A

Escherichia coli outer membrane phospholipase A (OMPLA) is an integral membrane enzyme. OMPLA is active as a homodimer and requires calcium as a cofactor. The crystal structures of the monomeric and the inhibited dimeric enzymes were recently determined [Snijder, H. J., et al. (1999) Nature 401, 717-721] and revealed that OMPLA monomers are folded into a 12-stranded antiparallel beta-barrel. The active site consists of previously identified essential residues Ser144 and His142 in an arrangement resembling the corresponding residues of a serine hydrolase catalytic triad. However, instead of an Asp or Glu that normally is present in the triad of serine hydrolases, a neutral asparagine (Asn156) was found in OMPLA. In this paper, the importance of the catalytic Asn156 is addressed by site-directed mutagenesis studies. All variants were purified at a 30 mg scale, and were shown to be properly folded using SDS-PAGE and circular dichroism spectroscopy. Using chemical cross-linking, it was shown that all variants were not affected in their calcium-dependent dimerization properties. The Asn156Asp variant exhibited a 2-fold lower activity than wild-type OMPLA at neutral pH. Interestingly, the activity of the variant is 1 order of magnitude higher than that of the wild type at pH >10. Modest residual activities (5 and 2.5%, respectively) were obtained for the Asn156Ala and Asn156Gln mutants, showing that the active site of OMPLA is more tolerant toward replacements of this third residue of the catalytic triad than other serine hydrolases, and that the serine and histidine residues are minimally required for catalysis. In the X-ray structure of dimeric OMPLA, the cofactor calcium is coordinating the putative oxyanion via two water molecules. We propose that this may lessen the importance for the asparagine in the catalytic triad of OMPLA.     

Open–closed conformational change revealed by the crystal structures of 3-keto-l-gulonate 6-phosphate decarboxylase from Streptococcus mutans

The 3-keto-l-gulonate 6-phosphate decarboxylase (KGPDC) catalyses the decarboxylation of 3-keto-l-gulonate 6-phosphate to l-xylulose in the presence of magnesium ions. The enzyme is involved in l-ascorbate metabolism and plays an essential role in the pathway of glucuronate interconversion. Crystal structures of Streptococcus mutans KGPDC were determined in the absence and presence of the product analog d-ribulose 5-phosphate. We have observed an 8 Å αB-helix movement and other structural rearrangements around the active site between the apo-structures and product analog bound structure. These drastic conformational changes upon ligand binding are the first observation of this kind for the KGPDC family. The flexibilities of both the α-helix lid and the side chains of Arg144 and Arg197 are associated with substrate binding and product releasing. The open–closed conformational changes of the active site, through the movements of the α-helix lid and the arginine residues are important for substrate binding and catalysis.     

Structures of Arg‐ and Gln‐type bacterial cysteine dioxygenase homologs

In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ～15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg‐type” enzymes) and some having a Gln substituted for this Arg (“Gln‐type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg‐type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln‐type” CDO homolog of uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron‐bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln‐type” CDO enzymes, we conclude that the “Gln‐type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3‐mercaptopropionate dioxygenases.     

Structure of the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii and its relation to other homotrimeric dUTPases

The bifunctional dCTP deaminase-dUTPase (DCD-DUT) from Methanocaldococcus jannaschii catalyzes the deamination of the cytosine moiety in dCTP and the hydrolysis of the triphosphate moiety forming dUMP, thereby preventing uracil from being incorporated into DNA. The crystal structure of DCD-DUT has been determined to 1.88-A resolution and represents the first known structure of an enzyme catalyzing dCTP deamination. The functional form of DCD-DUT is a homotrimer wherein the subunits are composed of a central distorted beta-barrel surrounded by two beta-sheets and four helices. The trimeric DCD-DUT shows structural similarity to trimeric dUTPases at the tertiary and quaternary levels. There are also additional structural elements in DCD-DUT compared with dUTPase because of a longer primary structure. Four of the five conserved sequence motifs that create the active sites in dUTPase are found in structurally equivalent positions in DCD-DUT. The last 25 C-terminal residues of the 204-residue-long DCD-DUT are not visible in the electron density map, but, analogous to dUTPases, the C terminus is probably ordered, closing the active site upon catalysis. Unlike other enzymes catalyzing the deamination of cytosine compounds, DCD-DUT is not exploiting an enzyme-bound metal ion such as zinc or iron for nucleophile generation. The active site contains two water molecules that are engaged in hydrogen bonds to the invariant residues Ser118, Arg122, Thr130, and Glu145. These water molecules are potential nucleophile candidates in the deamination reaction.     

New synthetic siderophores and their beta-lactam conjugates based on diamino acids and dipeptides

Linking of siderophores to antibiotics improves the penetration and therefore increases the antibacterial activity of the antibiotics. We synthesized the acylated catecholates and hydroxamates as siderophore components for antibiotic conjugates to reduce side effects of unprotected catecholate and hydroxamate moieties. In this paper, we report on bis- and tris-catecholates and mixed catecholate hydroxamates based on diamino acids or dipeptides. These compounds were active as siderophores in a growth promotion assay under iron limitation. Most of the conjugates with beta-lactams showed high in vitro activity against Gram-negative bacteria especially Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens and Stenotrophomonas maltophilia. The compounds with enhanced antibacterial activity use active iron uptake routes to penetrate the bacterial outer membrane barrier, demonstrated by assays with mutants deficient in components of the iron transport system. Correlation between chemical structure and biological activity was studied.     

Siderocalin/Lcn2/NGAL/24p3 Does Not Drive Apoptosis Through Gentisic Acid Mediated Iron Withdrawal in Hematopoietic Cell Lines

Siderocalin (also lipocalin 2, NGAL or 24p3) binds iron as complexes with specific siderophores, which are low molecular weight, ferric ion-specific chelators. In innate immunity, siderocalin slows the growth of infecting bacteria by sequestering bacterial ferric siderophores. Siderocalin also binds simple catechols, which can serve as siderophores in the damaged urinary tract. Siderocalin has also been proposed to alter cellular iron trafficking, for instance, driving apoptosis through iron efflux via BOCT. An endogenous siderophore composed of gentisic acid (2,5-dihydroxybenzoic acid) substituents was proposed to mediate cellular efflux. However, binding studies reported herein contradict the proposal that gentisic acid forms high-affinity ternary complexes with siderocalin and iron, or that gentisic acid can serve as an endogenous siderophore at neutral pH. We also demonstrate that siderocalin does not induce cellular iron efflux or stimulate apoptosis, questioning the role siderocalin plays in modulating iron metabolism.     

Siderophore production by Aeromonas salmonicida.

Growth under conditions of iron-restriction and the production of siderophores was examined in 21 typical and 14 atypical strains of Aeromonas salmonicida. With the exception of one atypical strain, all strains grew and multiplied in the presence of the high-affinity iron chelators ethylenediamine di(o-hydroxyphenylacetic acid), alpha, alpha'-dipyridyl or transferrin. Chrome azurol S agar was used to screen bacterial strains growing under these conditions for the production of siderophores. Siderophore production was detected only in the typical strains. Siderophores were also detected in the iron-restricted culture supernatants of typical strains. Siderophores were also detected in the iron-restricted culture supernatants of typical strains, where they were associated with an iron-binding activity. The siderophore was extracted from iron-restricted culture supernatant of one strain by adsorption onto an XAD-7 resin; it behaved as a 2,3-diphenol-catechol in several colorimetric assays. The results indicate that although both typical and atypical strains of A. salmonicida grow and multiply under conditions of iron-restriction, they use different iron-uptake mechanisms, siderophore-mediated and siderophore-independent, respectively. In cross-feeding assays, growth of typical strains was stimulated only by homologous iron-restricted supernatant, suggesting strain differences in the siderophore produced. However, one strain produced a culture supernatant with growth-stimulating activity for other typical and also atypical strains.