Iron-binding proteins and heme compounds as iron sources forVibrio anguillarum

Utilization of several iron sources available from the host was investigated in different strains ofVibrio anguillarum. We tested the ability to use transferrins, heme, hemoglobin, and haptoglobin-hemoglobin as iron sources in strains ofV. anguillarum possessing different iron uptake systems mediated by siderophores. Only the wild-type pathogenic strains with an intact siderophore-mediated iron transport system were able to obtain iron from transferrins. None of the low-virulence derivatives lacking siderophore production could grow in the presence of transferrins. However, all strains, wild-type and iron-deficient derivatives, could utilize heme, hemoglobin, and haptoglobin-hemoglobin as iron sources when added to iron-deficient media. The ability to grow in fish serum was also evaluated. Although only wild-type strains could grow in fresh serum, derivative strains lacking siderophore production also were able to grow when serum was heat inactivated or when a utilizable siderophore was present in serum. The results indicate that besides the siderophore-mediated mechanism,V. anguillarum can also obtain iron from other sources presumably available from the host, although its importance for growth in vivo is so far unknown.     

Growth of enterococci in normal and iron saturated serum]

Among 20 strains of genus Enterococcus isolated from different sources 12 strains multiplied in iron unsupplemented normal human serum. The excess of iron did not increase the rate of multiplication. Three strains survived in serum and growth of five strains was inhibited. The siderophores production had no effect on the multiplication of enterococci in serum unsupplemented with iron.     

Heme compounds as iron sources for nonpathogenic Rhizobium bacteria.

Many animal-pathogenic bacteria can use heme compounds as iron sources. Like these microorganisms, rhizobium strains interact with host organisms where heme compounds are available. Results presented in this paper indicate that the use of hemoglobin as an iron source is not restricted to animal-pathogenic microorganisms. We also demonstrate that heme, hemoglobin, and leghemoglobin can act as iron sources under iron-depleted conditions for Rhizobium meliloti 242. Analysis of iron acquisition mutant strains indicates that siderophore-, heme-, hemoglobin-, and leghemoglobin-mediated iron transport systems expressed by R. meliloti 242 share at least one component.     

Acquisition of iron by Trichodesmium and associated bacteria in culture

Trichodesmium colonies contain an abundant microbial consortium that is likely to play a role in nutrient cycling within the colony. This study used laboratory cultures of Trichodesmium and two genome-sequenced strains of bacteria typical of Trichodesmium-associated microbes to develop an understanding of the cycling of iron, a potentially limiting micronutrient, within Trichodesmium colonies. We found that the ferric siderophores desferrioxamine B and aerobactin were not readily bioavailable to Trichodesmium, relative to ferric chloride or citrate-associated iron. In contrast, the representative bacterial strains we studied were able to acquire iron from all of the iron sources, implying that naturally occurring Trichodesmium-associated bacteria may be capable of utilizing a more diverse array of iron sources than Trichodesmium. From the organism-specific uptake data collected in this study, a theoretical Trichodesmium colony was designed to model whole colony iron uptake. The bacteria accounted for most (>?70%) of the iron acquired by the colony, highlighting the importance of determining organism-specific uptake in a complex environment. Our findings suggest that, although they may share the same micro-environment, Trichodesmium and its colony-associated microbial cohort may differ substantially in terms of iron acquisition strategy.     

Responses of Saccharomyces cerevisiae Strains from Different Origins to Elevated Iron Concentrations

Iron is an essential micronutrient for all eukaryotic organisms. However, the low solubility of ferric iron has tremendously increased the prevalence of iron deficiency anemia, especially in women and children, with dramatic consequences. Baker''s yeast Saccharomyces cerevisiae is used as a model eukaryotic organism, a fermentative microorganism, and a feed supplement. In this report, we explore the genetic diversity of 123 wild and domestic strains of S. cerevisiae isolated from different geographical origins and sources to characterize how yeast cells respond to elevated iron concentrations in the environment. By using two different forms of iron, we selected and characterized both iron-sensitive and iron-resistant yeast strains. We observed that when the iron concentration in the medium increases, iron-sensitive strains accumulate iron more rapidly than iron-resistant isolates. We observed that, consistent with excess iron leading to oxidative stress, the redox state of iron-sensitive strains was more oxidized than that of iron-resistant strains. Growth assays in the presence of different oxidative reagents ruled out that this phenotype was due to alterations in the general oxidative stress protection machinery. It was noteworthy that iron-resistant strains were more sensitive to iron deficiency conditions than iron-sensitive strains, which suggests that adaptation to either high or low iron is detrimental for the opposite condition. An initial gene expression analysis suggested that alterations in iron homeostasis genes could contribute to the different responses of distant iron-sensitive and iron-resistant yeast strains to elevated environmental iron levels.     

Citric acid as a siderophore of enterococci?

In the pool of 70 enterococcal strains of the genus Enterococcus 61.4% released citrate into the medium. This metabolite has occurred more frequently in E. faecium strains. There was no correlation between hydroxamate siderophores production and citrate releasing. Only nine (10, 3%) of 70 strains have used Fe3+-dicitrate complex as iron sources. Iron restricted condition causing moderate inhibition of growth have not increased citrate releasing. When iron deficiency has caused stronger growth inhibition, E. faecalis strains did not release citrate and E. faecium strains its smaller amounts. The resting cells grown in iron-restricted condition have incorporated 59Fe3+ complexed by citrate more active than cells grown in the medium with excess of iron. So, citrate has not been a siderophore in enterococci.     

The effect of the nature of the iron sources on the growth of different Corynebacterium diphtheriae strains

In the study of the influence of organic and inorganic sources of iron on the growth of 5 C. diphtheriae clinical isolates bacterial growth was found to depended on the nature of the source of iron and its concentration. Differences between the strains in the level of growth, observed when ferric sulfate was used as the only source of iron in the medium, were established. Quantitative differences in the concentrations of inorganic and organic sources of iron, necessary for growth, were determined. The influence of three chemical chelators on the growth of C. diphtheriae under the conditions of iron deficiency in the culture medium was studied. The results of the study are indicative of the possibility of the differentiation of C. diphtheriae isolated according to the level of iron consumption.     

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.     

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.     

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.     

Evaluation of selected features of Staphylococcus cohnii enabling colonization of humans

Based on iron utilization, sensitivity to skin fatty acids, lipolytic and proteolytic activity the potential abilities ofStaphylococcus cohnii strains to colonize humans were evaluated. The investigation included 60 strains that belong to both subspecies,viz. S. cohnii ssp.cohnii andS. cohnii ssp.urealyticus. Strains were isolated from different sources of theIntensive Care Unit and from non-hospital environment. Most of the strains were multiple antibiotic-resistant. Strains of both subspecies revealed a relatively low iron requirement. These strains were capable of utilizing iron bound in oxo acids and from host iron-binding proteins.S. cohnii ssp.urealyticus were more effective in iron uptake thanS. cohnii ssp.cohnii. All investigated strains revealed sensitivity to skin fatty acids, butS. cohnii ssp.urealyticus strains were more resistant. Special features of strains of this subspecies promote colonization of humans.     

Production of transferrin receptors by Histophilus ovis: three of five strains require two signals

Five strains of Histophilus ovis (9L, 642A, 714, 5688T, and 3384Y) were investigated with respect to iron acquisition. All strains used ovine, bovine, and goat transferrins (Tfs), but not porcine or human Tfs, as iron sources for growth. In solid phase binding assays, total membranes from only two (9L and 642A) of the five strains, grown under iron-restricted conditions, were able to bind Tfs (ovine, bovine, and goat, but not porcine or human). However, when the organisms were grown under iron-restricted conditions in the presence of bovine transferrin (Tf), total membranes from all strains exhibited Tf binding (as above); competition experiments demonstrated that all three Tfs (ovine, bovine, and goat) were bound by the same receptor(s). Membranes from organisms grown under iron-replete conditions in the presence or absence of bovine Tf failed to bind any of the test Tfs. An affinity-isolation procedure allowed the isolation of two putative Tf-binding polypeptides (78 and 66 kDa) from total membranes of strains 9L and 642A grown under iron-restricted conditions, and from membranes of all strains if the growth medium also contained Tf. It is concluded that all strains tested acquire Tf-bound iron by means of siderophore-independent mechanisms involving surface receptors analogous to the Tf-binding proteins (TbpA and TbpB) found in comparable organisms; although iron restriction alone is sufficient to promote the expression of these proteins by strains 9L and 642A, their production by strains 714, 5688T, and 3384Y appears to require two signals, iron restriction and the presence of Tf.     

Assimilatory ferric reductases in enterococci

Enterococci produced assimilatory ferric reductases which are surface-associated enzymes. This is the first report of the intracellular enzymic reduction of iron by enterococci. A correlation between ferric reductases activity and species affiliation and origin of strains was found. The expression of ferric reductases has not affected by the presence or absence of iron, hemin and hemoglobin in the growth medium. Enterococcal ferric reductases exhibit a very broad specificity. A number of different ferric organic and inorganic compounds, natural and synthetic iron chelators and iron body sources including lactoferrin, transferin, ferritin, haemoglobin, could be reduced. A surface-associated ferric reductases may be one component of a general iron scavenging mechanism which can be used by enterococci growing in a variety of environments.     

Role of iron in the pathogenicity of Vibrio damsela for fish and mammals

Abstract The ability to obtain iron of 14 isolates of Vibrio damsela with different degrees of virulence for mice and turbot ( Scophthalmus maximus ) has been evaluated in artificial and natural iron-restricted environments. All strains were capable of utilizing haemoglobin (Hb) and ferric ammonium citrate (FAC) as the sole iron sources in vitro. However, only virulent V. damsela strains were able to resist the bacterioslatic and bactericidal effects of human and turbot sera, their growth being enhanced by the addition of Hb and FAC. The inhibitory effect of these sera on the growth of the non-pathogenic strain (ATCC 35083), however, was reversed by heat treatment (56°C for 60 min). The role of iron-availability on the virulence was investigated in iron-overloaded animals. The iron-treatment before the infection resulted in a significant reduction in the LD₅₀ of virulent strains. This fact demonstrates a positive correlation between iron availability in host fluids and degree of virulence in the species Vibrio damsela .     

The sensitivity of bacterial strains of Yersinia pestis isolated from voles to normal human serum]

The work deals with the results of the study of the viability of Y. pestis strains, isolated from voles in different natural foci of plague, in human normal serum (HNS) and its dependence on the assimilation of ions of exogenic iron. The cultures isolated at the Transcaucasian mountain focus of infection and having no small plasmid pYP were found highly sensitive to HNS. The introduction of the sources of iron, such as hemoglobin or ferritin, into the serum decreased its bactericidal effect.     

The glycolipid of Halobacterium saccharovorum

Abstract Of the five Bacteriodes species of the 'fragilis group' only Bacteroides fragilis was able to grow in human plasma. Therefore the capacity of several iron sources to stimulate to growth of Bacteroides species under iron restricted conditions in vitro was tested. The iron chelator bipyridyl was used for the restriction of iron in the media. Ferrous sulphate, ferric ammonium sulphate and ferric citrate stimulated the growth of all five Bacteroides species tested to the same extent. B. fragilis , and to a lesser extent B.thetaiotaomicron and B. distasonis were better able than B. vulgatus and B. ovatus to use haem-compounds as an iron source in the presence of the iron chelator bipyridyl. All five Bacteroides species tested could use 30% iron-saturated transferrin. There was no correlation between the ability of the strains to grow in human plasma and the ability to use either haem-compounds of transferrin as a source of iron.     

Differential expression of Bordetella pertussis iron transport system genes during infection

Temporal expression patterns of the Bordetella pertussis alcaligin, enterobactin and haem iron acquisition systems were examined using alcA-, bfeA- and bhuR-tnpR recombinase fusion strains in a mouse respiratory infection model. The iron systems were differentially expressed in vivo, showing early induction of the alcaligin and enterobactin siderophore systems, and delayed induction of the haem system in a manner consistent with predicted changes in host iron source availability during infection. Previous mixed infection competition studies established the importance of alcaligin and haem utilization for B. pertussis in vivo growth and survival. In this study, the contribution of the enterobactin system to the fitness of B. pertussis was confirmed using wild-type and enterobactin receptor mutant strains in similar competition infection experiments. As a correlate to the in vivo expression studies of B. pertussis iron systems in mice, sera from uninfected and B. pertussis-infected human donors were screened for antibody reactivity with Bordetella iron-repressible cell envelope proteins. Pertussis patient sera recognized multiple iron-repressible proteins including the known outer membrane receptors for alcaligin, enterobactin and haem, supporting the hypothesis that B. pertussis is iron-starved and responds to the presence of diverse iron sources during natural infection.     

Evaluation of novel assays to assess the influence of different iron sources on the growth of Clostridium difficile

The ability of four Clostridium difficile strains to utilize various exogenous organic and inorganic iron sources for growth under iron-depleted (250 μM DPP) and iron-limited (75 μM DPP) conditions was analyzed in liquid broth cultures grown in tubes and in microtiter plates, and data compared with results from a bioassay developed on solid media. The growth profile of C. difficile varied depending on the iron source and availability. Addition of FeSO(4), FeCl(3), Fe citrate and ferritin allowed growth in an iron-depleted environment whereas glycoproteins (iron-saturated and low-iron lactoferrin, apo- and holo-transferrin) and heme proteins (hemoglobin, hematin and hemin) did not. All iron sources, except lactoferrin, were able to restore bacterial growth under iron-limited conditions to varying extents. The results demonstrated that the broth microtiter assay developed here was reproducible, reliable and convenient for high-throughput analysis of the growth of C. difficile compared to alternative traditional methods.     

Virulence of Vibrio vulnificus: association with utilization of transferrin-bound iron, and lack of correlation with levels of cytotoxin or protease production

16 Vibrio vulnificus strains isolated from clinical and environmental sources were studied. 6 strains (4 clinical and 2 environmental) were virulent in both an infant mouse intragastric inoculation model and an iron-loaded adult mouse model; there was a close correlation between results in the two models. Virulence was not associated with increased in vitro production of extracellular cytotoxin-hemolysin or protease. There was some correlation between virulence and the ability of a strain to grow in an iron-limited medium, with a significant association observed between virulence and utilization of transferrin as an iron source. Our results suggest that the ability to make use of available host iron is an important determinant for pathogenicity of V. vulnificus.