Roles of Environmental Heme, and Menaquinone, in Streptococcus Agalactiae

Most bacteria require iron for growth. However, as it may not be directly available under aerobic conditions, bacteria may use iron-sequestering molecules, such as bacterially encoded siderophores, or heme, which is the major iron source in the animal host. Bacteria may also assimilate heme for purposes other than as an iron source. Once internalised, heme can activate, for example, a heme-dependent catalase and a cytochrome oxidase. In bacterial pathogen Streptococcus agalactiae, heme, in association with exogenous menaquinone, activates a respiratory chain. Respiration has radical effects on carbon metabolism. GBS respiration-grown cells display improved survival in an aerobic environment and greater virulence in a murine septicemia model. GBS might benefit from its ecological niches to capture heme and menaquinone, i.e., from other bacteria when it colonizes host mucosa, or from blood-containing organs during septicemia.     

Siderophore production by Enterobacter cloacae and a common receptor protein for the uptake of aerobactin and cloacin DF13.

Iron-starved cultures of Enterobacter cloacae produced two siderophores, identified as enterochelin and aerobactin. The aerobactin was excreted in larger amounts than was enterochelin, and it was synthesized preferentially in the late logarithmic and stationary growth phases under iron-deficient conditions. Enterochelin was synthesized by cultures in the logarithmic phase of growth and preferentially in medium with 1 microM ferric chloride. Both siderophores appeared to be excreted immediately after their synthesis, since no intracellular aerobactin or enterochelin could be detected. The killing activity of the bacteriocin cloacin DF13 was inhibited by aerobactin. It was shown that aerobactin and cloacin DF13 bound to the same receptor sites located in the outer membrane. The synthesis of these receptor sites was induced by iron limitation. We conclude that the receptor for the uptake of aerobactin also functions as receptor for cloacin DF13.     

Pseudomonas aeruginosa and its multiple strategies to access iron

Pseudomonas aeruginosa is a ubiquitous bacterium found in many natural and man-made environments. It is also a pathogen for plants, animals, and humans. As for almost all living organisms, iron is an essential nutrient for the growth of P. aeruginosa. The bacterium has evolved complex systems to access iron and maintain its homeostasis to survive in diverse natural and dynamic host environments. To access ferric iron, P. aeruginosa is able to produce two siderophores (pyoverdine and pyochelin), as well as use a variety of siderophores produced by other bacteria (mycobactins, enterobactin, ferrioxamine, ferrichrome, vibriobactin, aerobactin, rhizobactin and schizokinen). Furthermore, it can also use citrate, in addition to catecholamine neuromediators and plant-derived mono catechols, as siderophores. The P. aeruginosa genome also encodes three heme-uptake pathways (heme being an iron source) and one ferrous iron acquisition pathway. This review aims to summarize current knowledge concerning the molecular mechanisms involved in all the iron and heme acquisition strategies used by P. aeruginosa.     

Utilization of siderophores by Candida albicans

Candida albicans secretes both hydroxamate and phenolate-type siderophores when grown under iron-restricted conditions. The inhibition of candidal growth by iron limitation was reversed by the addition of supplemental hydroxamate on phenolate siderophores. Both siderophores produced equal stimulation of growth suggesting that C. albicans could utilize both siderophores with equal efficiency. Addition of heterologous siderophores from both bacteria and fungi also supported growth of the yeast in a deferrated medium. These results suggest that C. albicans has an iron-uptake mechanism which enables it to obtain iron by utilizing candidal and non-candidal siderophores.     

Recent insights into iron import by bacteria

Bacteria are confronted with a low availability of iron owing to its insolubility in the Fe3+ form or its being bound to host proteins. The bacteria cope with the iron deficiency by using host heme or siderophores synthesized by themselves or other microbes. In contrast to most other nutrients, iron compounds are tightly bound to proteins at the cell surfaces, from which they are further translocated by highly specific proteins across the cell wall of gram-positive bacteria and the outer membrane of gram-negative bacteria. Once heme and iron siderophores arrive at the cytoplasmic membrane, they are taken up across the cytoplasmic membrane by ABC transporters. Here we present an outline of bacterial heme and iron siderophore transport exemplified by a few selected cases in which recent progress in the understanding of the transport mechanisms has been achieved.     

Hydroxamate-siderophore production and utilization by marine eubacteria

Siderophore (iron-binding chelator) production was examined in 30 strains of open ocean bacteria from the generaVibrio, Alteromonas, Alcaligenes, Pseudomonas, andPhotobacterium. The results showed that hydroxamate-type siderophore production was widely distributed in various marine species, except for isolates ofAlteromonas macleodii andV. nereis. In all cases, the ability to produce siderophores was under the control of iron levels in the medium and satisfied the iron requirements of the siderophore bioassay organism. On the basis of chemical assay and bacterial bioassays, none of the examined isolates produced phenolate-type siderophores. Several isolates produces siderophores that were neither hydroxamatenor phenolate-type siderophores. Some strains such asAlteromonas communis produce siderophores that could be used by many other isolates. In contrast, the siderophore produced byAlcaligenes venustus had little cross-strain utilization. These findings suggest that the ability to produce siderophores may be common to open ocean bacteria.     

Control of the initial steps in heme biosynthesis in free-livingRhizobium sp. by culture conditions

Cell-free extracts obtained from free-livingRhizobium sp. in early stationary phase had three times as much 5-aminolevulinate synthase activity as did similar extracts from log phase cells. The level of 5-aminolevulinate dehydratase was also elevated at this point. The presence of 0.1 mM hemin in the culture medium prevented the transitory increase in enzyme activities during this early stationary phase. The effect of hemin was counteracted by 1 mg bovine serum albumin per milliliter medium. This control of the development of 5-aminolevulinate synthase and 5-aminolevulinate dehydratase activities by free hemin suggests a mechanism by which heme and globin formation might be coordinated for the synthesis of leghemoglobin in legume root nodules.     

Siderophore-mediated iron acquisition in the staphylococci

Iron is frequently a growth-limiting nutrient due to its propensity to interact with oxygen to form insoluble precipitates and, therefore, biological systems have evolved specialized uptake mechanisms to obtain this essential nutrient. Many pathogenic bacteria are capable of obtaining stringently sequestered iron from animal hosts by one or both of the following mechanisms: extraction of heme from host erythrocyte and serum hemoproteins, or through the use of high affinity, iron-scavenging molecules termed siderophores. This review summarizes our current knowledge of siderophore-mediated iron acquisition systems in the genus Staphylococcus.     

Induction of stolon settlement in the scyphopolyps ofAurelia aurita (Cnidaria,Scyphozoa, Semaeostomeae) by glycolipids of marine bacteria

The settlement of pedal stolons of scyphopolyps ofAurelia aurita Lamarck could be induced by addition of a species of bacteria from the family Micrococcaceae. After treatment of the bacteria with several organic solvents a crude lipid extract free of bacteria could be obtained which was shown to be effective in inducing stolon settlement. Crude lipids extracted from the late logarithmic growth phase had an optimal effect on stolon attachment, in contrast to previously published experiments showing that all logarithmic phases of bacteria had the same level of effectiveness. After separation of the crude lipid extracts by thin layer chromatography and subsequent bioassay of the reeluated substances, acylgalactosidyldiglyceride and monogalactosidyldiglyceride were identified as the effective substances. Monogalactosidyldiglyceride was only found in bacteria from the medium logarithmic growth phase, whereas the former was found at all stages. The effectiveness of acylgalactosidyldiglyceride was independent of the growth phase of the extracted bacteria.     

The Fe Chelator Proferrorosamine A Is Essential for the Siderophore-Mediated Uptake of Iron by Pseudomonas roseus fluorescens

Pseudomonas roseus fluorescens produces, besides the Fe chelator proferrorosamine A, Fe -chelating compounds, called siderophores. The production of proferrorosamine A and siderophores by P. roseus fluorescens appears to be controlled in a similar way by the concentration of available iron and by the concentration of dissolved oxygen. The higher the concentration of iron available for the microorganism, the lower the production of both chelating compounds. However, the production of siderophores was much more sensitive to iron availability than was proferrorosamine A production. Proferrorosamine A and siderophores were only produced in minimal medium C if the concentration of dissolved oxygen ranged from 4.5 to 2.0 ppm. At higher or lower concentrations, none of the iron-chelating compounds were produced. Furthermore, it has been shown that proferrorosamine-negative Tn5 mutants of P. roseus fluorescens were able to form siderophores only under iron-limiting conditions when proferrorosamine A was added to the medium. Our data suggest that proferrorosamine A production is essential for siderophore synthesis by P. roseus fluorescens; the production of siderophores occurred only when proferrorosamine A was present.     

Iron uptake and metabolism in pseudomonads

Pseudomonads are ubiquitous Gram-negative γ proteobacteria known for their extreme versatility and adaptability. Some are plant pathogens (Pseudomonas syringae) which have to survive on the surface of leaves while others can colonize the rhizosphere or survive in soil (Pseudomonas fluorescens, Pseudomonas putida), and one species, Pseudomonas entomophila, is an insect pathogen. The most investigated species, Pseudomonas aeruginosa, is known to be an opportunistic pathogen able to infect plants, nematodes, insects, and mammals, including humans. Like for other bacteria, iron is a key nutrient for pseudomonads. The fluorescent pseudomonads produce siderophores, the best known being the fluorescent high-affinity peptidic pyoverdines. Often diverse secondary siderophores of lower affinity are produced as well (pyochelin, pseudomonin, corrugatins and ornicorrugatins, yersiniabactin, and thioquinolobactin). Reflecting their large capacity of adaptation to changing environment and niche colonization, pseudomonads are able to obtain their iron from heme or from siderophores produced by other microorganisms (xenosiderophores) via the expression of outer membrane TonB-dependent receptors. As expected, iron uptake is exquisitely and hierarchically regulated in these bacteria. In this short review, the diversity of siderophores produced, receptors, and finally the way iron homeostasis is regulated in P. aeruginosa, P. syringae, P. putida, and P. fluorescens, will be presented and, when possible, put in relation with the lifestyle and the ecological niche.     

Identification of a siderophore utilization locus in nontypeable <Emphasis Type="Italic">Haemophilus influenzae</Emphasis>

Background  

Haemophilus influenzae has an absolute aerobic growth requirement for either heme, or iron in the presence of protoporphyrin IX. Both iron and heme in the mammalian host are strictly limited in their availability to invading microorganisms. Many bacterial species overcome iron limitation in their environment by the synthesis and secretion of small iron binding molecules termed siderophores, which bind iron and deliver it into the bacterial cell via specific siderophore receptor proteins on the bacterial cell surface. There are currently no reports of siderophore production or utilization by H. influenzae.     

A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus

Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD–I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis.     

Catecholate Siderophores Protect Bacteria from Pyochelin Toxicity

Background

Bacteria produce small molecule iron chelators, known as siderophores, to facilitate the acquisition of iron from the environment. The synthesis of more than one siderophore and the production of multiple siderophore uptake systems by a single bacterial species are common place. The selective advantages conferred by the multiplicity of siderophore synthesis remains poorly understood. However, there is growing evidence suggesting that siderophores may have other physiological roles besides their involvement in iron acquisition.

Methods and Principal Findings

Here we provide the first report that pyochelin displays antibiotic activity against some bacterial strains. Observation of differential sensitivity to pyochelin against a panel of bacteria provided the first indications that catecholate siderophores, produced by some bacteria, may have roles other than iron acquisition. A pattern emerged where only those strains able to make catecholate-type siderophores were resistant to pyochelin. We were able to associate pyochelin resistance to catecholate production by showing that pyochelin-resistant Escherichia coli became sensitive when biosynthesis of its catecholate siderophore enterobactin was impaired. As expected, supplementation with enterobactin conferred pyochelin resistance to the entE mutant. We observed that pyochelin-induced growth inhibition was independent of iron availability and was prevented by addition of the reducing agent ascorbic acid or by anaerobic incubation. Addition of pyochelin to E. coli increased the levels of reactive oxygen species (ROS) while addition of ascorbic acid or enterobactin reduced them. In contrast, addition of the carboxylate-type siderophore, citrate, did not prevent pyochelin-induced ROS increases and their associated toxicity.

Conclusions

We have shown that the catecholate siderophore enterobactin protects E. coli against the toxic effects of pyochelin by reducing ROS. Thus, it appears that catecholate siderophores can behave as protectors of oxidative stress. These results support the idea that siderophores can have physiological roles aside from those in iron acquisition.     

Iron uptake mechanisms as key virulence factors in bacterial fish pathogens

This review summarizes the current knowledge about iron uptake systems in bacterial fish pathogens and their involvement in the infective process. Like most animal pathogens, fish pathogens have evolved sophisticated iron uptake mechanisms some of which are key virulence factors for colonization of the host. Among these systems, siderophore production and heme uptake systems are the best studied in fish pathogenic bacteria. Siderophores like anguibactin or piscibactin, have been described in Vibrio and Photobacterium pathogens as key virulence factors to cause disease in fish. In many other bacterial fish pathogens production of siderophores was demonstrated but the compounds were not yet chemically characterized and their role in virulence was not determined. The role of heme uptake in virulence was not yet clearly elucidated in fish pathogens although there exist evidence that these systems are expressed in fish tissues during infection. The relationship of other systems, like Fe(II) transporters or the use of citrate as iron carrier, with virulence is also unclear. Future trends of research on all these iron uptake mechanisms in bacterial fish pathogens are also discussed.     

The Fe2+ Chelator Proferrorosamine A Is Essential for the Siderophore-Mediated Uptake of Iron by Pseudomonas roseus fluorescens

Pseudomonas roseus fluorescens produces, besides the Fe²⁺ chelator proferrorosamine A, Fe³⁺ -chelating compounds, called siderophores. The production of proferrorosamine A and siderophores by P. roseus fluorescens appears to be controlled in a similar way by the concentration of available iron and by the concentration of dissolved oxygen. The higher the concentration of iron available for the microorganism, the lower the production of both chelating compounds. However, the production of siderophores was much more sensitive to iron availability than was proferrorosamine A production. Proferrorosamine A and siderophores were only produced in minimal medium C if the concentration of dissolved oxygen ranged from 4.5 to 2.0 ppm. At higher or lower concentrations, none of the iron-chelating compounds were produced. Furthermore, it has been shown that proferrorosamine-negative Tn5 mutants of P. roseus fluorescens were able to form siderophores only under iron-limiting conditions when proferrorosamine A was added to the medium. Our data suggest that proferrorosamine A production is essential for siderophore synthesis by P. roseus fluorescens; the production of siderophores occurred only when proferrorosamine A was present.     

Cell Size,Macromolecule Composition,Nuclear Number,Oxygen Consumption and Cyst Formation During Two Growth Phases in Unagitated Cultures of Acanthamoeba castellanii1

SYNOPSIS. The size, composition and physiology of average cells have been studied in cultures of Acanthamoeba castellanii during the phases of logarithmic growth and population growth deceleration (PGD). Most of the features examined were relatively constant during log phase, but had significant changes during PGD. Average cell volume increased about 60% and total dry mass about 15–20% during the latter period. Total protein content remained constant thruout both growth phases, but cytochrome oxidase doubled during PGD. DNA, RNA and glycogen levels began to change during late log phase. DNA decreased about 50% and RNA increased about 75%. Glycogen decreased 50% during the RNA build-up and then increased to a plateau above the log phase level. A final decrease in glycogen followed an increase in the relative numbers of cysts in late PGD. It was found that PGD begins when O₂ becomes limiting and evidence that the subsequent changes in macromolecule composition are related to encystation is discussed.     

Iron requirement and siderophore production in Rhizobium ciceri during growth on an iron-deficient medium

Under conditions of iron limitation many rhizospheric bacteria produce siderophores, ferric iron-specific ligands, which may enhance plant growth by increasing the availability of iron near the roots. Thirty-five strains of Rhizobium ciceri, specific to chickpea (Cicer arietinum L.), were screened for their ability to grow on iron-deficient medium and to produce siderophores. Maximal growth of all strains previously depleted in iron was obtained in medium containing 5 to 10 m of ferric iron. When iron limitation was achieved by the addition of 2,2-bipyridyl or EDDHA [ethylene diamine di(o-hydroxyphenyl) acetic acid] to the medium, only two strains were able to scavenge iron and grow. Siderophore production by these two strains was detected by the Chrome Azurol S assay (CAS), a universal test for siderophores. No hydroxamate-type siderophores were detected in the supernatants of Rhizobium ciceri cultures. However, some strains secreted salicylic acid and 2,3-dihydroxybenzoic acid as phenolate-type siderophores. Addition of ferric iron to the culture medium increased growth yield significantly but depressed the production of siderophores. Although these compounds are produced in response to iron deficiency, nutritive components of the culture medium significantly affected their production. It seems that CuII, MoVI and MnII ions bound competitively with iron to siderophores, resulting in a 34 to 100% increase in production.     

NUCLEOLAR AND BIOCHEMICAL CHANGES DURING UNBALANCED GROWTH OF TETRAHYMENA PYRIFORMIS

Numerous nucleoli can be observed in the macronucleus of the logarithmically growing ciliated protozoan Tetrahymena pyriformis; at late log phase the nucleoli aggregate and fuse. In stationary phase this fusion process continues, leaving a very few large vacuolated nuclear fusion bodies in the nucleus. When these stationary phase cells are placed into fresh enriched proteose peptone medium, the large fusion bodies begin to disaggregate during the 2.5-hour lag phase before cell division is initiated. By 3 to 6 hours after inoculation the appearance of the nucleoli in many cells returns to what it was in logarithmic cells. In view of the possible role of nucleoli in ribosome synthesis, attempts were made to correlate the morphological changes to changes in RNA and protein metabolism. The beginning of an increased RNA synthesis was concomitant with the beginning of disaggregation of the large fusion bodies into nucleoli, which was noticed in some cells by 1 hour after the return to fresh enriched proteose peptone medium. Increased protein synthesis then followed the increased RNA synthesis by 1 hour. The supply of RNA precursors (essential pyrimidines) were removed from cultures which were grown on a chemically defined synthetic medium, in order to study the relation between nucleolar fusion and synthesis of RNA and protein. Pyrimidine deprivation drastically curtailed RNA and protein synthesis, but did not cause fusion of nucleoli. When pyrimidines were added back to this culture medium, RNA synthesis was immediately stimulated and again preceded an increased protein synthesis by 1 hour. These studies suggest the involvement of unfused nucleoli in RNA and protein synthesis and demonstrate the extreme plasticity of nucleoli with respect to changes in their environment.     

Kinetics of biosynthesis of iron-regulated membrane proteins in Escherichia coli

Using biological iron chelators to control specifically iron availability to Escherichia coli K-12 in conjunction with radioactive pulse-labels, we examined the biosynthesis of six iron-regulated membrane proteins. Iron deprivation induced the synthesis of five proteins, which had molecular weights of 83,000 (83K), 81K (Fep), 78K (TonA), 74K (Cir), and 25K. The kinetics of induction were the same in entA and entA⁺ strains, but were affected by the initial iron availability in the media. Iron-poor cells induced rapidly (half-time, 10 min), whereas iron-rich cells began induction after a lag and showed a slower induction half-time (30 min). Within this general pattern of induction after iron deprivation, several different kinetic patterns were apparent. The 83K, 81K, and 74K proteins were coordinately controlled under all of the conditions examined. The 78K and 25K proteins were regulated differently. The synthesis of a previously unrecognized 90K inner membrane protein was inhibited by iron deprivation and stimulated by iron repletion. Both ferrichrome and ferric enterobactin completely repressed 81K and 74K synthesis when the siderophores were supplied at concentrations of 5 μM in vivo (half-time, 2.5 min). At concentrations less than 5 μM, however, both siderophores repressed synthesis only temporarily; the duration of repression was proportional to the amount of ferric siderophore added. The half-lives of the 81K and 74K mRNAs, as measured by rifampin treatment, were 1.2 and 1.6 min, respectively. The results of this study suggest that enteric bacteria are capable of instantaneously detecting and reacting to fluctuations in the extracellular iron concentration and that they store iron during periods of iron repletion for utilization during periods of iron stress. Neither iron storage nor iron regulation of envelope protein synthesis is dependent on the ability of the bacteria to form heme.