Iron Uptake Mechanisms in the Fish Pathogen Tenacibaculum maritimum

We present here the first evidence of the presence of iron uptake mechanisms in the bacterial fish pathogen Tenacibaculum maritimum. Representative strains of this species, with different serotypes and origins, were examined. All of them were able to grow in the presence of the chelating agent ethylenediamine-di- (o-hydroxyphenyl acetic acid) (EDDHA) and also produced siderophores. Cross-feeding assays suggest that the siderophores produced are closely related. In addition, all T. maritimum strains utilized transferrin, hemin, hemoglobin, and ferric ammonic citrate as iron sources when added to iron-deficient media. Whole cells of all T. maritimum strains, grown under iron-supplemented or iron-restricted conditions, were able to bind hemin, indicating the existence of constitutive binding components located at the T. maritimum cell surface. This was confirmed by the observation that isolated total and outer membrane proteins from all of the strains, regardless of the iron levels of the media, were able to bind hemin, with the outer membranes showing the strongest binding. proteinase K treatment of whole cells did not affect the hemin binding, indicating that, in addition to proteins, some protease-resistant components could also bind hemin. At least three outer membrane proteins were induced in iron-limiting conditions, and all strains, regardless of their serotype, showed a similar pattern of induced proteins. The results of the present study suggest that T. maritimum possesses at least two different systems of iron acquisition: one involving the synthesis of siderophores and another that allows the utilization of heme groups as iron sources by direct binding.     

Binding of haemin by the fish pathogen Photobacterium damselae subsp. piscicida

Whole cells of virulent (DI 21 and B 51) and avirulent (ATCC 29690 and EPOY 8803-II) strains of Photobacterium damselae subsp. piscicida, grown under iron-supplemented or iron-restricted conditions, were able to bind haemin. Iron limitation resulted in an increased binding of haemin by DI 21, B 51 and ATCC 29690 cells but did not affect the haemin-binding ability of the EPOY 8803-II cells. Proteinase K treatment of whole cells markedly reduced the binding of haemin, indicating that protein receptors located at the cell surface are involved in the binding. This was confirmed by the observation that isolated total as well as outer membrane proteins from all the strains, regardless of the iron levels of the media, were able to bind haemin, with the outer membranes showing the strongest binding. Haemin binding by membrane protein extracts was not affected by heat treatment but was almost completely abolished by Proteinase K treatment, suggesting the presence of thermostable protein receptors for haemin. The capsular polysaccharide also appears to play a minor role in binding of haemin. It was concluded that constitutive as well as inducible mechanisms of haemin binding occur in P. damselae subsp. piscicida. These mechanisms would rely mainly upon the direct interaction between the haemin molecules and surface-exposed outer membrane protein receptors.     

TonB or not TonB: is that the question?

Bacteria are able to survive in low-iron environments by sequestering this metal ion from iron-containing proteins and other biomolecules such as transferrin, lactoferrin, heme, hemoglobin, or other heme-containing proteins. In addition, many bacteria secrete specific low molecular weight iron chelators termed siderophores. These iron sources are transported into the Gram-negative bacterial cell through an outer membrane receptor, a periplasmic binding protein (PBP), and an inner membrane ATP-binding cassette (ABC) transporter. In different strains the outer membrane receptors can bind and transport ferric siderophores, heme, or Fe3+ as well as vitamin B12, nickel complexes, and carbohydrates. The energy that is required for the active transport of these substrates through the outer membrane receptor is provided by the TonB/ExbB/ExbD complex, which is located in the cytoplasmic membrane. In this minireview, we will briefly examine the three-dimensional structure of TonB and the current models for the mechanism of TonB-dependent energy transduction. Additionally, the role of TonB in colicin transport will be discussed.     

Genetic variability of the heme uptake system among different strains of the fish pathogen Vibrio anguillarum: identification of a new heme receptor

The ability to utilize heme compounds as iron sources was investigated in Vibrio anguillarum strains belonging to serotypes O1 to O10. All strains, regardless of their serotype or isolation origin could utilize hemin and hemoglobin as sole iron sources. Similarly, all of the isolates could bind hemin and Congo red, and this binding was mediated by cell envelope proteins. PCR and Southern hybridization were used to assay the occurrence of heme transport genes huvABCD, which have been previously described in serotype O1. Of 23 strains studied, two serotype O3 isolates proved negative for all huvABCD genes, whereas nine strains included in serotypes O2, O3, O4, O6, O7, and O10 tested negative for the outer membrane heme receptor gene huvA. A gene coding for a novel outer membrane heme receptor was cloned and characterized in a V. anguillarum serotype O3 strain lacking huvA. The new heme receptor, named HuvS, showed significant similarity to other outer membrane heme receptors described in Vibrionaceae, but little homology (39%) to HuvA. This heme receptor was present in 9 out of 11 of the V. anguillarum strains that tested negative for HuvA. Furthermore, complementation experiments demonstrated that HuvS could substitute for the HuvA function in Escherichia coli and V. anguillarum mutants. The huvS and huvA sequences alignment, as well as the analysis of their respective upstream and downstream DNA sequences, suggest that horizontal transfer and recombination might be responsible for generating this genetic diversity.     

Identification of iron-regulated outer membrane proteins in uropathogenic Proteus mirabilis and its relationship with heme uptake

The effect of iron deprivation on the expression of outer membrane proteins and the ability to use heme as an iron source by uropathogenic Proteus mirabilis , Pr 6515, was studied. Examination of iron-restricted bacteria showed three outer membrane proteins ranging from 66 to 75 kDa to be affected by iron restriction, as well as a newly expressed 64-kDa protein. These proteins were induced within 15 minutes of iron-deprivation. The strain grew in the presence of ferric citrate, hemin and hemoglobin as iron sources, but could not use transferrin, lactoferrin or siderophores from exogenous sources. The 64- and 66-kDa proteins showed hemin-binding activity by affinity chromatography, and both reacted in Western blots with sera from mice transurethrally infected with the same strain. We suggest that P. mirabilis expresses iron-regulated outer membrane proteins that could be involved in heme uptake and may have a role in pathogenesis.     

Evidence that Yersinia ruckeri possesses a high affinity iron uptake system.

This work represents the first evidence of the presence of an iron uptake system siderophore mediated in the bacterial fish pathogen Yersinia ruckeri. A group of 20 strains representative of this species, with different serotype and origin were examined. All of them were able to grow at high concentrations (from 0.7 to 1.1 mM) of the iron chelator EDDA. Although the Y. ruckeri isolates failed to cross-feed the indicator strains for enterobactin and aerobactin production, the chemical tests revealed the presence in the culture supernatants of phenolate siderophores. At least three outer membrane proteins were induced in iron limiting conditions. All the strains showed a similar pattern of induced membrane proteins regardless their serotype or origin, which suggests a similarity in the iron uptake system. This system could have an important role in the pathogeneicity of Y. ruckeri for fish.     

Genetic Variability of the Heme Uptake System among Different Strains of the Fish Pathogen Vibrio anguillarum: Identification of a New Heme Receptor

The ability to utilize heme compounds as iron sources was investigated in Vibrio anguillarum strains belonging to serotypes O1 to O10. All strains, regardless of their serotype or isolation origin could utilize hemin and hemoglobin as sole iron sources. Similarly, all of the isolates could bind hemin and Congo red, and this binding was mediated by cell envelope proteins. PCR and Southern hybridization were used to assay the occurrence of heme transport genes huvABCD, which have been previously described in serotype O1. Of 23 strains studied, two serotype O3 isolates proved negative for all huvABCD genes, whereas nine strains included in serotypes O2, O3, O4, O6, O7, and O10 tested negative for the outer membrane heme receptor gene huvA. A gene coding for a novel outer membrane heme receptor was cloned and characterized in a V. anguillarum serotype O3 strain lacking huvA. The new heme receptor, named HuvS, showed significant similarity to other outer membrane heme receptors described in Vibrionaceae, but little homology (39%) to HuvA. This heme receptor was present in 9 out of 11 of the V. anguillarum strains that tested negative for HuvA. Furthermore, complementation experiments demonstrated that HuvS could substitute for the HuvA function in Escherichia coli and V. anguillarum mutants. The huvS and huvA sequences alignment, as well as the analysis of their respective upstream and downstream DNA sequences, suggest that horizontal transfer and recombination might be responsible for generating this genetic diversity.     

Iron acquisition in the dental pathogen Actinobacillus actinomycetemcomitans: What does it use as a source and how does it get this essential metal?

Actinobacillus actinomycetemcomitans requires iron to grow under limiting conditions imposed by synthetic and natural chelators. Although none of the strains tested used hemoglobin, lactoferrin or transferrin, all of them used FeCl₃ and hemin as iron sources under chelated conditions. Dot-blot binding assays showed that all strains bind lactoferrin, hemoglobin, and hemin but not transferrin. When compared with smooth strains, the rough isolates showed higher hemin binding activity, which was sensitive to proteinase K treatment. A. actinomycetemcomitans harbors the Fur-regulated afeABCD locus coding for iron acquisition in isogenic and non-isogenic cell backgrounds. The genome of this oral pathogen also harbors several other predicted iron uptake genes including the hitABC locus, which restored iron acquisition in the E. coli 1017 ent mutant. However, the disruption of this locus in the parental strain did not affect iron acquisition as drastically as the inactivation of AfeABCD, suggesting that the latter system could be more involved in iron transport than the HitABC system. The genome of this oral pathogen also harbors an active copy of the exbBexbDtonB operon, which could provide the energy needed for hemin acquisition. However, inactivation of each coding region of this operon did not affect the hemin and iron acquisition phenotypes of isogenic derivatives. This observation suggests that the function of these proteins could be replaced by those coded for by tolQ, tolR and tolA as it was described for other bacterial transport systems. Interruption of a hasR homolog, an actively transcribed gene that is predicted to code for an outer membrane hemophore receptor protein, did not affect the ability of an isogenic derivative to bind and use hemin under chelated conditions. This result also indicates that A. actinomycetemcomitans could produce more than one outer membrane hemin receptor as it was described in other human pathogens. All strains tested formed biofilms on plastic under iron-rich and iron-chelated conditions. However, smooth strains attached poorly and formed weaker biofilms when compared with rough isolates. The incubation of rough cells in the presence of FeCl₃ or hemin resulted in an increased number of smaller aggregates and microcolonies as compared to the fewer but larger aggregates formed when cells were grown in the presence of dipyridyl.     

Structural biology of bacterial iron uptake

To fulfill their nutritional requirement for iron, bacteria utilize various iron sources which include the host proteins transferrin and lactoferrin, heme, and low molecular weight iron chelators termed siderophores. The iron sources are transported into the Gram-negative bacterial cell via specific uptake pathways which include an outer membrane receptor, a periplasmic binding protein (PBP), and an inner membrane ATP-binding cassette (ABC) transporter. Over the past two decades, structures for the proteins involved in bacterial iron uptake have not only been solved, but their functions have begun to be understood at the molecular level. However, the elucidation of the three dimensional structures of all components of the iron uptake pathways is currently limited. Despite the low sequence homology between different bacterial species, the available three-dimensional structures of homologous proteins are strikingly similar. Examination of the current three-dimensional structures of the outer membrane receptors, PBPs, and ABC transporters provides an overview of the structural biology of iron uptake in bacteria.     

Structural biology of bacterial iron uptake

To fulfill their nutritional requirement for iron, bacteria utilize various iron sources which include the host proteins transferrin and lactoferrin, heme, and low molecular weight iron chelators termed siderophores. The iron sources are transported into the Gram-negative bacterial cell via specific uptake pathways which include an outer membrane receptor, a periplasmic binding protein (PBP), and an inner membrane ATP-binding cassette (ABC) transporter. Over the past two decades, structures for the proteins involved in bacterial iron uptake have not only been solved, but their functions have begun to be understood at the molecular level. However, the elucidation of the three dimensional structures of all components of the iron uptake pathways is currently limited. Despite the low sequence homology between different bacterial species, the available three-dimensional structures of homologous proteins are strikingly similar. Examination of the current three-dimensional structures of the outer membrane receptors, PBPs, and ABC transporters provides an overview of the structural biology of iron uptake in bacteria.     

Evidence that Yersinia ruckeri possesses a high affinity iron uptake system

Abstract: This work represents the first evidence of the presence of an iron uptake system siderophore mediated in the bacterial fish pathogen Yersinia ruckeri . A group of 20 strains representative of this species, with different serotype and origin were examined. All of them were able to grow at high concentrations (from 0.7 to 1.1 mM) of the iron chelator EDDA. Although the Y. ruckeri isolates failed to cross-feed the indicator strains for enterobactin and aerobactin production, the chemical tests revealed the presence in the culture supernatants of phenolate siderophores. At least three outer membrane proteins were induced in iron limiting conditions. All the strains showed a similar pattern of induced membrane proteins regardless their serotype or origin, which suggests a similarity in the iron uptake system. This system could have an important role in the pathogenicity of Y. ruckeri for fish.     

Phenotypic characterization and description of two major O-serotypes in Tenacibaculum maritimum strains from marine fishes

Tenacibaculum maritimum is the etiological agent of marine flexibacteriosis disease, with the potential to cause severe mortalities in various cultured marine fishes. The development of effective preventive measures (i.e. vaccination) requires biochemical, serological and genetic knowledge of the pathogen. With this aim, the biochemical and antigenic characteristics of T. maritimum strains isolated from sole, turbot and gilthead sea bream were analysed. Rabbit antisera were prepared against sole and turbot strains to examine the antigenic relationships between the 29 isolates and 3 reference strains. The results of the slide agglutination test, dot-blot assay and immunoblotting of lipopolysaccharides (LPS) and membrane proteins were evaluated. All bacteria studied were biochemically identical to the T. maritimum reference strains. The slide agglutination assays using O-antigens revealed cross-reaction for all strains regardless of the host species and serum employed. However, when the dot-blot assays were performed, the existence of antigenic heterogeneity was demonstrated. This heterogeneity was supported by immunoblot analysis of the LPS, which clearly revealed 2 major serological groups that were distinguishable without the use of absorbed antiserum: Serotypes O1 and O2. These 2 serotypes seem to be host-specfic. In addition, 2 sole isolates and the Japanese reference strains displayed cross-reaction with both sera in all serological assays, and are considered to constitute a minor serotype, O1/O2. Analysis of total and outer membrane proteins revealed that all strains share a considerable number of common bands that are antigenically related.     

Effects of host iron transport compounds on growth kinetics and outer-membrane protein expression of Bilophila wadsworthia

Since the environmental iron concentration has emerged as an important attribute in the expression of bacterial virulence, the purpose of this study was to determine the effects of transferrin, lactoferrin, heme compounds, and inorganic iron sources (ferric and ferrous sulfate) on the growth of Bilophila wadsworthia and to study its outer membrane composition when grown under these different simulated in vivo conditions. Lactoferrin, transferrin, hemin and hemoglobin supported full growth of the bacteria in media lacking other iron sources. Bilophila wadsworthia was also capable of growing in the presence of ferrous and ferric sulfate. Profiles obtained by SDS-PAGE showed two iron-regulated outer membrane proteins (IROMPs) of 190 kDa and 88 kDa. The 190 kDa was susceptible to proteinase K cleavage in whole cells, indicating its exposure at the cell surface. These two major IROMPs were expressed in iron-restricted media supplemented with iron-bound organic sources and repressed by the addition of inorganic iron sources.     

Novel hemin binding domains in the Corynebacterium diphtheriae HtaA protein interact with hemoglobin and are critical for heme iron utilization by HtaA

The human pathogen Corynebacterium diphtheriae utilizes hemin and hemoglobin as iron sources for growth in iron-depleted environments. The use of hemin iron in C. diphtheriae involves the dtxR- and iron-regulated hmu hemin uptake locus, which encodes an ABC hemin transporter, and the surface-anchored hemin binding proteins HtaA and HtaB. Sequence analysis of HtaA and HtaB identified a conserved region (CR) of approximately 150 amino acids that is duplicated in HtaA and present in a single copy in HtaB. The two conserved regions in HtaA, designated CR1 and CR2, were used to construct glutathione S-transferase (GST) fusion proteins (GST-CR1 and GST-CR2) to assess hemin binding by UV-visual spectroscopy. These studies showed that both domains were able to bind hemin, suggesting that the conserved sequences are responsible for the hemin binding property previously ascribed to HtaA. HtaA and the CR2 domain were also shown to be able to bind hemoglobin (Hb) by the use of an enzyme-linked immunosorbent assay (ELISA) method in which Hb was immobilized on a microtiter plate. The CR1 domain exhibited a weak interaction with Hb in the ELISA system, while HtaB showed no significant binding to Hb. Competitive binding studies demonstrated that soluble hemin and Hb were able to inhibit the binding of HtaA and the CR domains to immobilized Hb. Moreover, HtaA was unable to bind to Hb from which the hemin had been chemically removed. Alignment of the amino acid sequences of CR domains from various Corynebacterium species revealed several conserved residues, including two highly conserved tyrosine (Y) residues and one histidine (H) residue. Site-directed mutagenesis studies showed that Y361 and H412 were critical for the binding to hemin and Hb by the CR2 domain. Biological assays showed that Y361 was essential for the hemin iron utilization function of HtaA. Hemin transfer experiments demonstrated that HtaA was able to acquire hemin from Hb and that hemin bound to HtaA could be transferred to HtaB. These findings are consistent with a proposed mechanism of hemin uptake in C. diphtheriae in which hemin is initially obtained from Hb by HtaA and then transferred between surface-anchored proteins, with hemin ultimately transported into the cytosol by an ABC transporter.     

Identification of heme-binding proteins in the cell membranes of Vibrio anguillarum

Abstract Two strains of Vibrio anguillarum belonging to O1 and O2 serotypes were examined for their ability to bind hemin and hemoglobin. Whole cells as well as membrane extracts from both strains could clearly bind hemin and hemoglobin constitutively. Hemoglobin binding was completely inhibited by a 100-fold excess of unlabelled hemoglobin and also by hemin, suggesting the existence of specific receptors for heme groups in the cell membranes. Several hemin-binding and hemoglobin-binding bands with similar molecular sizes were detected in polyacrylamide gels as well as in Western blots. Only two of these protein bands in both strains were iron-regulated while the others were independent of the cell iron status. We conclude that both serotypes of V. anguillarum possess heme-binding abilities by means of membrane proteins.     

Outer membrane proteome of Prevotella intermedia 17: identification of thioredoxin and iron-repressible hemin uptake loci

Although hemin is an indispensable nutrient for the oral pathogen Prevotella intermedia, not much is known regarding the molecular mechanisms of hemin acquisition. The availability of the genomic sequence of the bacterium allowed us to apply proteomic approaches to identify proteins that may be mediating the hemin acquisition process. As hemin acquisition mechanisms have been shown to be induced in iron-depleted conditions, we applied proteomic approaches to detect those proteins whose expressions were affected by iron. We analyzed 40 protein spots and identified 19 such proteins. Interestingly, two proteins drastically upregulated in iron-depleted conditions, PIN0009 and PINA0611, are homologs of hemin uptake receptors in other bacteria. PIN0009 is predicted to be an outer membrane lipoprotein. It is encoded by a gene that is the first of a seven-gene genomic locus encoding proteins of a novel hemin acquisition system. The second protein, PINA0611, is a homolog of numerous TonB-dependent outer membrane receptors including outer membrane iron uptake receptors of various Gram-negative bacteria. There was also another protein, regulated by iron, that was previously demonstrated to bind hemoglobin in P. intermedia. Finally, we identified a thioredoxin-like protein that has a novel outer membrane location.     

A genetic locus involved in iron utilization unique to some Campylobacter strains.

Two genes involved in iron utilization in Campylobacter coli VC167 T1 have been characterized. The cfrA gene encodes a protein with a predicted Mr of 77,653 which, after processing of the leader sequence, has a predicted Mr of 75,635. This protein has significant sequence identity to siderophore receptors of several bacteria, and site-specific mutants defective in cfrA do not synthesize one of two major iron-repressible outer membrane proteins. An adjacent gene encodes a TonB-like protein; a mutant in this gene lost the ability to utilize hemin, ferrichrome, and enterochelin as iron sources. The cfrA and tonB genes of VC167 T1 hybridized to all strains of C. coli and most strains of C. jejuni examined but did not hybridize to several other strains of C. jejuni, suggesting that the thermophilic campylobacters can be separated into two categories based on the presence of these two iron utilization genes.     

Siderophore-Mediated Iron Sequestering by Shewanella putrefaciens

The iron-sequestering abilities of 51 strains of Shewanella putrefaciens isolated from different sources (fish, water, and warm-blooded animals) were assessed. Thirty strains (60%) produced siderophores in heat-sterilized fish juice as determined by the chrome-azurol-S assay. All cultures were negative for the catechol-type siderophore, whereas 24 of the 30 siderophore-producing strains tested positive in the Csáky test, indicating the production of siderophores of the hydroxamate type. Siderophore-producing S. putrefaciens could to some degree cross-feed on the siderophores of other S. putrefaciens strains and on compounds produced by an Aeromonas salmonicida strain under iron-limited conditions. The siderophores of S. putrefaciens were not sufficiently strong to inhibit growth of other bacteria under iron-restricted conditions. However, siderophore-producing Pseudomonas bacteria were always inhibitory to S. putrefaciens under iron-limited conditions. Growth of siderophore-producing strains under iron-limited conditions induced the formation of one major new outer membrane protein of approximately 72 kDa. Two outer membrane proteins of approximately 53 and 23 kDa were not seen when iron was restricted.