Iron-regulated Outer Membrane Proteins Research Articles

Iron restriction and the growth ofSalmonella enteritidis

Strains of Salmonella enteritidis were examined for their ability to remove ferric-ions from the iron chelating agents ovotransferrin, Desferal and EDDA. Growth of S. enteritidis phage type (PT) 4 (SE4) in trypticase soy broth containing ovotransferrin resulted in the expression of iron regulated outer membrane proteins (OMPs) of 74, 78 and 81 kDa, and unexpectedly the repression of expression of OMP C. The 38 MDa 'mouse virulence' plasmid was not required for the expression of the iron-regulated OMPs (IROMPs). SE4 was able to obtain iron bound to the iron chelator Desferal and EDDA without expressing a high-affinity iron uptake system. Strains of S. enteritidis belonging to PTs 7, 8, 13a, 23, 24 and 30 were also able to remove ferric ions from Desferal and EDDA without expressing a high-affinity iron uptake system. We conclude that strains of SE4 possess a high-affinity iron sequestering mechanism that can readily remove iron from ovotransferrin. It is likely that iron limitation, and not iron restriction, is responsible for the bacteriostatic properties of fresh egg whites.

Zinc affects siderophore-mediated high affinity iron uptake systems in the rhizosphere Pseudomonas aeruginosa 7NSK2.

Zinc concentrations ranging between 0.1 and 1 mM only slightly reduced maximal growth of wild-type Pseudomonas aeruginosa 7NSK2 in iron-limiting casamino acid medium, but had a clear negative effect on the growth of mutant MPFM1 (pyoverdin negative) and especially mutant KMPCH (pyoverdin and pyochelin negative). Production of pyoverdin by wild-type strain 7NSK2 was significantly increased in the presence of 0.5 mM zinc and could not be repressed by iron even at a concentration of 100 microM. Siderophore detection via isoelectrofocusing revealed that mutant KMPCH did not produce any siderophores, while mutant MPFM1 overproduced a siderophore with an acidic isoelectric point, most likely pyochelin. Pyochelin production by MPFM1 was stimulated by the presence of zinc in a similar way as pyoverdin for the wild-type. Analysis of outer membrane proteins revealed that three iron regulated outer membrane proteins (IROMPs) (90, 85 and 75 kDa) were induced by iron deficiency in the wild-type, while mutants were found to have altered IROMP profiles. Zinc specifically enhanced the production of a 85 kDa IROMP in 7NSK2, a 75 kDa IROMP in MPFM1 and a 90 kDa IROMP in KMPCH.

Growth promotion of synthetic catecholate derivatives on Gram-negative bacteria

Derivatives of benzoic acid, glyoxylic acid benzhydrazone, oxanilic acid and N-dihydroxybenzylidene-2,4,6-trimethylaminobenzene were investigated as catecholic iron chelators under iron-depleted conditions. Some of the compounds showed strong positive reactions in the universal chemical siderophore assay (CAS): 3,4-dihydroxybenzoic acid, glyoxylic acid 2,3-dihydroxybenzhydrazone, N-3,4-dihydroxybenzylidene-2,4,6-trimethylaminobenzene. In particular these compounds also enabled removal of iron from iron-saturated transferrin. Using various siderophore indicator strains (enterobacteriacecae, Pseudomonas aeruginosa and Aeromonas hydrophila mutants) in bioassays the following growth promotion could be detected: vicinal substituents (e.g. 2,3- or 3,4-) were essential, the carboxyamido group seen in benzoic acids and glyoxylic acid benzhydrazones contributed to a positive reaction as well as the azomethin group (in N-3,4-dihydroxybenzylidene-2,4,6-trimethylaminobenzene). 2,3-Dihydroxybenzoic acid and the 2,3-diacetoxy substitute preferably promoted growth of enterobacteriaceae mutants. In contrast, the 3,4- positioned compounds preferably promoted growth of P. aeruginosa mutants and A. hydrophila SB 22. Glyoxylic acid di(methoxycarbonyloxy)-benzhydrazones (2,3- and 3,4- positioned) including the 2,3-dihydroxy compound preferably enabled growth of the non-fermenters. N-3,4-dihydroxybenzylidene-2,4,6-trimethylaminobenzene supplied all mutants of Salmonella, Escherichia coli, Klebsiella, Morganella, P. aeruginosa and A. hydrophila with iron. Transport of glyoxylic acid 2,3-dihydroxybenzhydrazone depended on tonB, and required the involvement of the iron-regulated outer membrane proteins (IROMPs) FepA, Cir and Fiu.

Siderophores and related outer membrane proteins produced by pseudomonads isolated from eels and freshwater

A total of 46 environmental pseudomonads, together with six type strains, were examined for their siderophore-producing activity. All strains were able to grow under iron-limiting conditions, gave orange halos in the CAS agar assay, and produced hydroxamates, and some of them also produced phenolate-type compounds. Bioassays showed that all strains, except Pseudomonas aeruginosa, promoted growth of mutant strain Arthrobacter flavescens JG-9, deficient in hydroxamate production, and some of them promoted growth of Salmonella typhimurium enb-1, which requires enterobactin for growth. The presence of iron-regulated outer membrane proteins was observed, the molecular size of the main induced proteins ranged between 76 and 93 kDa.

Siderophores and related outer membrane proteins produced by pseudomonads isolated from eels and freshwater.

A total of 46 environmental pseudomonads, together with six type strains, were examined for their siderophore-producing activity. All strains were able to grow under iron-limiting conditions, gave orange halos in the CAS agar assay, and produced hydroxamates, and some of them also produced phenolate-type compounds. Bioassays showed that all strains, except Pseudomonas aeruginosa, promoted growth of mutant strain Arthrobacter flavescens JG-9, deficient in hydroxamate production, and some of them promoted growth of Salmonella typhimurium enb-1, which requires enterobactin for growth. The presence of iron-regulated outer membrane proteins was observed, the molecular size of the main induced proteins ranged between 76 and 93 kDa.

Siderophore-specific induction of iron uptake in Pseudomonas aeruginosa.

Pseudomonas aeruginosa has two siderophore-based high-affinity iron-uptake systems utilizing pyoverdin and pyochelin. Using strain IA1, a mutant deficient in production of both siderophores, we have shown that addition of purified siderophore to the growth medium induces expression of specific iron-regulated outer-membrane proteins and increases 55Fe-siderophore transport. Addition of pyoverdin from the parent strain PAO1 or from a clinical strain 0:12 induced expression of an 85 kDa IROMP and increased the rate of 55Fe-pyoverdin transport. Transport rates for 55Fe-PAO1 pyoverdin increased from 1.27 to 3.57 pmol Fe min-1 per 10(9) cells. Addition of purified pyochelin induced expression of a 75 kDa IROMP accompanied with increased 55Fe-pyochelin uptake without affecting 55Fe-pyoverdin transport. 55Fe-pyochelin transport increased from 0.3 to 10.6 pmol min-1 per 10(9) cells. Addition of pyoverdin from the parent strain or a chromatographically distinct pyoverdin caused increased reactivity with an anti-85 kDa mAb in Western blotting, indicating that the same receptor is being induced. These results suggest that P. aeruginosa can respond specifically to the presence of siderophore and moreover that not only can the pyoverdin receptor transport its cognate ferri-pyoverdin but also different ferri-pyoverdins, albeit at a reduced rate.

Survival and root colonization of mutants of plant growth‐promoting pseudomonads affected in siderophore biosynthesis or regulation of siderophore production

Abstract The importance of the high affinity iron‐uptake system for survival and root colonization was studied via mutants of plant growth‐promoting pseudomonads affected in siderophore biosynthesis or regulation of siderophore production. In a nutrient‐enriched soil, but not in a normal soil, survival of regulatory and biosynthetic siderophore mutants was impaired in comparison with the wild type strain. Especially a mutation in an apparent “stress”; regulation of siderophore production had a deleterious effect on survival. Biosynthetic side mutants colonized maize roots as well as the parent strains. However, the root colonization capabilities of a mutant affected in the iron regulation of siderophore production were significantly impaired, probably because this mutant is unable to induce ironregulated outer membrane proteins (IROMPs) for its own siderophores and those produced by other microorganisms under iron‐limiting conditions. The results presented suggest that competition for iron is more importa...

Iron acquisition and hemolysin production by Campylobacter jejuni

Campylobacter jejuni strains were tested for their ability to acquire iron from various iron sources present in humans. Hemin, hemoglobin, hemin-hemopexin, and hemoglobin-haptoglobin stimulated the growth of C. jejuni strains in low-iron medium. Transferrin, lactoferrin, and ferritin were unable to provide iron to the strains tested. Derivatives of the naturally transformable C. jejuni strain 81-176 were isolated on the basis of their inability to use hemin as an iron source. These mutants were also unable to use hemoglobin, hemin-hemopexin, or hemoglobin-haptoglobin as iron sources. Some mutants lacked a 71,000-Da iron-regulated outer membrane protein, while others appeared to retain all of their outer membrane proteins. Growth curves and a recombination experiment that exploited natural transformation were used to further characterize the mutants. A hemolytic activity was shown to be produced by several C. jejuni strains, but it did not appear to be iron regulated.

Metal regulation of siderophore synthesis in Pseudomonas aeruginosa and functional effects of siderophore-metal complexes.

Pseudomonas aeruginosa synthesizes two siderophores, pyochelin and pyoverdin, characterized by widely different structures, physicochemical properties, and affinities for Fe(III). Titration experiments showed that pyochelin, which is endowed with a relatively low affinity for Fe(III), binds other transition metals, such as Cu(II), Co(II), Mo(VI), and Ni(II), with appreciable affinity. In line with these observations, Fe(III) and Co(II) at 10 microM or Mo(VI), Ni(II), and Cu(II) at 100 microM repressed pyochelin synthesis and reduced expression of iron-regulated outer membrane proteins of 75, 68, and 14 kDa. In contrast, pyoverdin synthesis and expression of the 80-kDa receptor protein were affected only by Fe(III). All of the metals tested, except Mo(VI), significantly promoted P. aeruginosa growth in metal-poor medium; Mo(VI), Ni(II), and Co(II) were more efficient as pyochelin complexes than the free metal ions and the siderophore. The observed correlation between the affinity of pyochelin for Fe(III), Co(II), and Mo(VI) and the functional effects of these metals indicates that pyochelin may play a role in their delivery to P. aeruginosa.

Modulation of surface antigen expression by Klebsiella pneumoniae in response to growth environment

Growth in pooled human body fluids [urine, serum and peritoneal dialysate (HPD)] modulated the expression of cell envelope antigens in virulent (serotype O1:K1) and avirulent (serotype O1:K66) Klebsiella pneumoniae strains. Marked variations in the outer membrane protein (OMP) and lipopolysaccharide (LPS) profiles were noted when broth-grown cells were compared with those of bacteria cultured in body fluids. In particular, for the O1:K1 serotype strain, growth in the latter resulted in: (a) the expression of at least five iron-regulated OMPs in the 74–87 kDa range, the pattern of which was medium dependent; (b) alterations in the migration of the LPS core polysaccharide; and (c) the reversion of isogenic O −:K + and O −:K − mutants to the O + phenotype after growth in fresh serum but not in heat-inactivated serum, urine or HPD. Similar results were obtained for the O1:K66 serotype, although no variation in the migration of the LPS core was noted. For both O1:K1 and O1:K66 serotypes, neither the surface exposure of O1 serotype LPS nor the production of K-antigen (capsular polysaccharide) was affected by growth in body fluids. No reversion of K − mutants to the K + phenotype was observed. These data illustrate the phenotypic flexibility of this opportunistic pathogen and emphasise the crucial role of the O − rather than the K-antigen in protecting K. pneumoniae from complement-mediated serum killing.

Characterization of a Vibrio cholerae virulence factor homologous to the family of TonB-dependent proteins.

IrgA is an iron-regulated virulence factor for infection in an animal model with classical Vibrio cholerae strain 0395. We detected gene sequences hybridizing to irgA at high stringency in clinical isolates in addition to 0395, including another classical strain of V. cholerae, three V. cholerae strains of the El Tor biotype, three non-O1 isolates of V. cholerae, and individual isolates of Vibrio parahaemolyticus, Vibrio fluvialis, and Vibrio alginolyticus. No hybridization to irgA was seen with chromosomal DNA from Vibrio vulnificus or Aeromonas hydrophila. To verify that irgA is the structural gene for the major iron-regulated outer membrane protein of V. cholerae, we determined the amino-terminal sequence of this protein recovered after gel electrophoresis and demonstrated that it corresponds to the amino acid sequence of IrgA deduced from the nucleotide sequence. Gel electrophoresis showed that two El Tor strains of V. cholerae had a major iron-regulated outer membrane protein identical in size and appearance to IrgA in strain 0395, consistent with the findings of DNA hybridization. We have previously suggested that IrgA might be the outer membrane receptor for the V. cholerae siderophore, vibriobactin. Biological data presented here, however, show that a mutation in irgA had no effect on the transport of vibriobactin and produced no defect in the utilization of iron from ferrichrome, ferric citrate, haemin or haemoglobin. The complete deduced amino acid sequence of IrgA demonstrated homology to the entire class of Escherichia coli TonB-dependent proteins, particularly Cir. Unlike the situation with Cir, however, we were unable to demonstrate a role for IrgA as a receptor for catechol-substituted cephalosporins. The role of IrgA in the pathogenesis of V. cholerae infection, its function as an outer membrane receptor, and its potential interaction with a TonB-like protein in V. cholerae remain to be determined.

The pyocin Sa receptor of Pseudomonas aeruginosa is associated with ferripyoverdin uptake.

We have used Tn5 mutagenesis to obtain a mutant resistant to pyocin Sa. When grown in iron-deficient succinate medium this mutant lacked an 85-kDa iron-regulated outer membrane protein (IROMP), and expression of a 75-kDa IROMP was increased compared with that in the parent strain. The mutant was deficient in pyoverdin biosynthesis and showed a 95% decrease in transport of ferripyoverdin purified from the parent strain, suggesting that the 85-kDa IROMP is the specific receptor for ferripyoverdin and pyocin Sa. The mutant compensated for the deficiency in pyoverdin biosynthesis and transport by exhibiting a fourfold increase in ferripyochelin transport. The low-level transport of ferripyoverdin in the Sa-resistant mutant, which extended to heterologous pyoverdins from other strains, suggests that Pseudomonas aeruginosa has a second ferripyoverdin uptake system of lower affinity and broader specificity.

Immunogenicity and antigenic heterogeneity of a human transferrin-binding protein in Neisseria meningitidis.

Growing Neisseria meningitidis on an iron restriction medium induces the synthesis of new outer membrane proteins, some of them true iron-regulated outer membrane proteins (IROMPs) and others synthesized because of the stress produced by the iron restriction. Some of these proteins are antigenic and can be considered for the development of vaccines; this is especially desirable in the case of N. meningitidis serogroup B, for which polysaccharide vaccines are not efficient. The antigenicity of N. meningitidis 37- and 70-kDa IROMPs has been studied previously; in this work, we studied the immunogenicity and antigenic heterogeneity of another IROMP, the human transferrin-binding protein 2 (TBP2), which seems to be indispensable for meningococcal growth inside the host. Mice were inoculated with purified outer membrane vesicles (blebs) from 5 selected N. meningitidis strains, and the five serum samples obtained were analyzed for anti-TBP2 antibodies by using the homologous strain and for cross-reactivity with the TBP2 of the 4 other selected strains and another 35 heterologous N. meningitidis strains. The TBP2s of the 5 strains tested were all immunogenic in mice to various degrees depending on the strain, and all five TBP2s shared one or more epitopes with heterologous strains (as shown by the cross-reactivities of the five serum samples), although the number of cross-reacting strains was very variable, ranging from 2 for strain V002 to 35 for strain P391. This suggests that the TBP2 epitopes of different strains differ in nature or in their accessibility to the immune system. Under the iron restriction conditions used, all strains synthesized a non-TBP2 antigenic 56-kDa protein thought to be a stress protein.

Correlation between the Ability of Haemophilus paragallinarum to Acquire Ovotransferrin-Bound Iron and the Expression of Ovotransferrin-Specific Receptors

To investigate the mechanisms of iron acquisition in avian haemophili, strains of Haemophilus paragallinarum and H. avium were tested for siderophore production and utilization of transferrin iron for growth. No evidence of siderophore production was detected in either of these species using a functional screening assay. H. paragallinarum, but not strains of H. avium, was able to acquire iron from 30% saturated chicken and turkey transferrins but not from human, porcine, or bovine transferrins. In response to iron limitation, H. paragallinarum expressed four iron-regulated outer-membrane proteins of 53, 62, 66, and 94 kilodaltons (kDa). Only the 53- and 94-kDa proteins were detected in the H. avium strains. Using affinity methods, the 94- and 53-kDa proteins were isolated specifically by chicken or turkey transferrin, indicating that they may be equivalent to transferrin binding proteins (TBP1 and TBP2, respectively) isolated from other bacterial species. The isolation of the 62- and 66-kDa proteins in association with TBP1 and TBP2 under less stringent washing conditions only in H. paragallinarum implicates these proteins in the iron acquisition process.

Cloning, sequencing, and transcriptional regulation of viuA, the gene encoding the ferric vibriobactin receptor of Vibrio cholerae.

A 74-kDa iron-regulated outer membrane protein of Vibrio cholerae acts as the receptor for the V. cholerae iron-siderophore complex, ferric vibriobactin. MBG14, a mutant of V. cholerae 0395 containing a TnphoA insertion in a gene designated viuA, lacks this 74-kDa outer membrane protein and is unable to bind or utilize exogenous ferric vibriobactin. Introduction of a plasmid containing the complete viuA coding sequence and 513 bp of upstream DNA into MBG14 restored ferric vibriobactin utilization to the mutant. The DNA insert in this plasmid was sequenced, revealing a single open reading frame of 2,061 bp, encoding a deduced protein of 687 amino acids with a predicted molecular mass of 76,417 Da and a predicted initial signal sequence of 37 amino acids. ViuA showed only weak homology to two iron-regulated outer membrane proteins in Escherichia coli, IutA and FecA. Construction of viuA::TnphoA gene fusions allowed study of the regulation of viuA expression by iron. This regulation in E. coli was dependent on the fur gene. Northern (RNA) blot analysis of RNA from wild-type V. cholerae grown in high- and low-iron media revealed a monocistronic viuA message that was negatively regulated by iron at the transcriptional level. Primer extension analysis identified a single transcriptional start site, located 243 bp above the translational start site. The promoter region of viuA contained two interrupted dyad symmetric nucleotide sequences, overlapping the -10 and -35 boxes, each similar to the E. coli Fur binding consensus sequence. Another iron-regulated gene in V. cholerae that is negatively regulated by fur, irgA, requires a positive transcriptional activator (irgB) for expression. However, a strain of V. cholerae mutant in irgB was unaffected in viuA expression. These studies suggest that there is conserved, global coordinate iron regulation in V. cholerae by fur; additional regulatory factors, superimposed upon the fur system, may provide more precise control of individual iron-regulated genes.

Identification of the vibriobactin receptor of Vibrio cholerae.

Vibrio cholerae produces the novel phenolate siderophore vibriobactin and several outer membrane proteins in response to iron starvation. To determine whether any of these iron-regulated outer membrane proteins serves as the receptor for vibriobactin, the classical V. cholerae strain 0395 was mutagenized by using TnphoA, and iron-regulated fusions were analyzed for vibriobactin transport. One mutant, MBG14, was unable to bind or utilize exogenous vibriobactin and did not grow in low-iron medium. However, synthesis of the siderophore and transport of other iron complexes, including ferrichrome, hemin, and ferric citrate, were unaffected in MBG14. Analysis of membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the loss from the mutant of a 74-kDa iron-regulated outer membrane protein present in the parental strain when grown in iron-limiting conditions. This protein partitioned into the detergent phase during Triton X-114 extraction, suggesting that it is a hydrophobic membrane protein. DNA sequences encoding the gene into which TnphoA had inserted, designated viuA (vibriobactin uptake), restored the wild-type phenotype to the mutant; the complemented mutant expressed the 74-kDa outer membrane protein under iron-limiting conditions and possessed normal vibriobactin binding and uptake. These data indicate that the 74-kDa outer membrane protein of V. cholerae serves as the vibriobactin receptor.

In vivo expression of iron regulated outer-membrane proteins in Pasteurella haemolytica-A1

Pasteurella haemolytica-A1 was grown in vitro under iron-rich conditions, iron-depleted conditions, and in vivo within a chamber implanted in the peritoneal cavity of a rabbit to determine if iron regulated outer-membrane proteins were expressed in vivo. The antigenicity of outer membrane (OM) proteins from bacteria grown under these conditions was assessed by immunoblotting with pooled serum from convalescent bovine calves experimentally infected with P. haemolytica-A1 and serum from the implanted rabbit. Pasteurella haemolytica-A1 grown under iron-depleted conditions showed three distinct OM protein bands (71, 77, and 100 kDa) that were present in much lesser amounts when the organism was grown under iron-rich conditions. These same three bands were evident in OM protein preparations from bacteria grown in vivo. Western blotting indicated that these protein bands were recognized immunologically by the convalescent bovine serum and by serum from the implanted rabbit, in cells grown under the iron-depleted conditions and in vivo, but not if the bacteria were grown under the in vitro iron-rich conditions.

Growth characteristics and expression of iron-regulated outer-membrane proteins of chemostat-grown biofilm cells of Pseudomonas aeruginosa

An in vitro chemostat system was used to study the growth and the expression of iron-regulated outer-membrane proteins (IROMPs) by biofilm cells of Pseudomonas aeruginosa cultivated under conditions of iron limitation. The population of the planktonic cells decreased when the dilution rate was increased. At a dilution rate of 0.05 h-1, the populations of planktonic cells of both mucoid and nonmucoid P. aeruginosa were 3 x 10(9) cells/mL. This value dropped to 5 x 10(6) cells/mL when the dilution rate was increased to 1.0 h-1. The reverse was observed for the biofilm cells. The number of biofilm cells colonising the silicone tubing increased when the dilution rate was increased. The number of biofilm cells of the mucoid strain at steady state was 2 x 10(8) cells/cm (length) when the dilution rate was fixed at 0.05 h-1. The figure increased to 8 x 10(9) cells/cm when the dilution rate was increased to 1.0 h-1. The population of biofilm cells of the nonmucoid strain was 9 x 10(7) cells/cm (length) when the dilution rate was 0.05 h-1. It increased to 2 x 10(9) cells/cm when the dilution rate was set at 1.0 h-1. The expression of IROMPs was induced in the biofilm cells of both mucoid and nonmucoid strains when the dilution rates were 0.05 and 0.2 h-1. IROMPs were reduced but still detectable at the dilution rate of 0.5 h-1. However, the expression of IROMPs was repressed when the dilution rate was increased to 1.0 h-1. The data suggest that the biofilm cells of P. aeruginosa switch on the expression of IROMPs to assist iron acquisition when the dilution rate used for the chemostat run is below 0.5 h-1. The high affinity iron uptake system is not required by the biofilm cells when the dilution rate is increased because the trace amount of iron present in the chemostat is sufficient for the growth of adherent biofilm cells.

Pyochelin-mediated iron transport in Pseudomonas aeruginosa: involvement of a high-molecular-mass outer membrane protein

An iron-regulated outer membrane protein of 75,000 daltons was strongly expressed following iron limitation of strains of Pseudomonas aeruginosa which fail to produce pyoverdine. A mutant nonderepressible for this protein (K372) was deficient in pyochelin-mediated iron transport at 150 nM FeCl3, consistent with a role for the 75-kDa protein in ferripyochelin transport. Moreover, ferripyochelin specifically protected the 75-kDa protein against trypsin digestion, supporting an interaction between ferripyochelin and the 75-kDa protein. Previous reports implicated a 14,000-dalton outer membrane protein as the receptor for ferripyochelin (P.A. Sokol and D.E. Woods, Infect. Immun. 40:665-669, 1983) and demonstrated that a mutant (FBP-28) expressing a defective 14-kDa outer membrane protein did not exhibit pyochelin-mediated iron transport (P.A. Sokol, J. Bacteriol. 169:3365-3368, 1987). Nonetheless, we were able to demonstrate (i) that FBP-28 was inducible for the 75-kDa protein under iron-limiting conditions and (ii) that concomitant with the induction of this protein in FBP-28, pyochelin-mediated iron uptake at 150 nM FeCl3 was observed. Interestingly, strain K372 did transport ferripyochelin at higher (750 nM) FeCl3 concentrations, suggesting that a second pyochelin-mediated iron transport system, perhaps involving the 14-kDa outer membrane protein identified previously, operates in P. aeruginosa.

Yersinia ruckeri produces four iron-regulated outer membrane proteins but does not produce detectable siderophores

Abstract. The expression of iron-regulated outer membrane proteins (OMPs) was examined in 36 isolates of Yersinia ruckeri selected to represent a range of biotypes, serotypes and OMP-types. Iron-restricted growth was achieved in tryptone soya broth by chelation with 200 μm α,α'-dipyridyl, although similar results could be achieved with 25 μm transferrin or 280 μM ethylenediaminedihydroxyphenylacetic acid (EDDA). During growth under iron-restricted conditions, all 36 isolates produced four additional outer membrane proteins of molecular weights 72, 69·5, 68 and 66 kDa. Expression of these proteins was repressed by the addition of 100 μm FeCl3 to the growth medium, confirming that the proteins were iron-regulated. The facts that all of the isolates examined possessed the same four iron-regulated OMPs, and that the plasmid content of different serotypes is known to vary indicated that production of these proteins is probably chromosomally mediated and not plasmid-mediated. Production of siderophores was not detected in low iron media and suggested a direct iron-uptake mechanism for this organism.