Iron transport in Mycobacterium smegmatis: occurrence of iron-regulated envelope proteins as potential receptors for iron uptake

Cell-envelope fractions were isolated from the rapidly growing saprophyte Mycobacterium smegmatis following growth in glycerol/asparagine medium under both iron-limited (0.02 microgram Fe ml-1) and iron-sufficient (2.0 to 4.0 micrograms Fe ml-1) conditions. Examination of these preparations by SDS-PAGE demonstrated the production of at least four additional proteins when iron was limiting. These iron-regulated envelope proteins (IREPs) were ascribed apparent molecular masses of 180 kDa (protein I), 84 kDa (protein II), 29 kDa (protein III) and 25 kDa (protein IV). All four proteins were present in both cell-wall and membrane preparations but spheroplast preparations were devoid of the 29 kDa protein. Attempts at labelling the proteins with 55FeCl3 or 55Fe-exochelin, the siderophore for iron uptake, were unsuccessful, though this was attributed to the denatured state of the proteins following electrophoresis. Antibodies were raised to each of the four proteins: the one raised to protein III inhibited exochelin-mediated iron uptake into iron-deficiently grown cells by 70% but was ineffective against iron uptake into iron-sufficiently grown cells. As exochelin is taken up into both types of cells by a similar process, protein III may not be a simple receptor for iron uptake though the results imply some function connected with this process. The role of the other IREPs is less certain.     

Iron uptake from ferric citrate by Vibrio anguillarum

We report here that Vibrio anguillarum possesses a non-inducible active transport system which can efficiently supply iron to the cell from ferric citrate, independently of the siderophore-based mechanisms. The strains tested were able to grow in CM9 medium in iron-restricted conditions when ferric citrate was present in the medium. Moreover, the presence of ferric citrate inhibited the production of siderophores in the strains tested. V. anguillarum cells and isolated membranes could incorporate ⁵⁵Fe³⁺ complexed by citrate, without a difference between cells grown in the presence or absence of ferric citrate. The presence of 2,4-dinitrophenol, ferrozine, ferricyanide, trypsin, as well as low temperature produced a marked decrease or total inhibition of ⁵⁵Fe³⁺ uptake by the cells. All these results suggest that iron uptake from ferric citrate in V. anguillarum must be an energy-dependent process not induced by the presence of iron or citrate in the medium, mediated by a membrane protein(s), which may require an iron reduction step to function.     

Amino acid transport in Mycobacterium smegmatis

The transport of d-alanine, d-glutamic acid, and d-valine in Mycobacterium smegmatis was compared quantitatively with that of their l-isomers. It appeared that the uptake of d-alanine was mediated by an active process displaying saturation kinetics characteristic of enzyme function, whereas the uptake of d-glutamic acid was accomplished by a passive process showing diffusion kinetics. Both processes were involved in the uptake of l-alanine, l-glutamic acid, d-valine, and l-valine. d-Valine competed with l-valine for entry into the cell through a single active process. d-Alanine and l-alanine also utilized the same active process, but the d-isomer could not enter the cell through the passive process. The passive process exhibited characteristics of diffusion, but was sensitive to sulfhydryl-blocking reagents and showed competition among structurally related amino acids. These last findings suggested that the passive process is a facilitated diffusion.     

Amylooligosaccharides from Mycobacterium smegmatis.

In early stationary phase of growth, Mycobacterium smegmatis cultures accumulate amylooligosaccharides (alpha 1 leads to 4-glucooligosaccharides) up to the undecasaccharide. Although M. smegmatis also makes an acylated polymethylpolysaccharide that is predominantly and alpha 1 leads to 4-glucan, we conclude that these oligosaccharides are precursors of glycogen rather than lopopolysaccharide.     

Uptake of iron by apoferritin from a ferric dihydrolipoate complex

A study was made on the uptake of iron by horse spleen apoferritin, by using as an iron source the same ferric dihydrolipoate complex which represents the major product in the anaerobic removal of ferritin-bound iron by dihydrolipoate at neutral pH. The ferric dihydrolipoate complex was chemically synthesized and used as an iron donor to apoferritin. Iron uptake was studied, at slightly alkaline pH and in anaerobic conditions, as a function of the concentration of both the iron donor and apoferritin. Isolation of ferritin from mixtures of ferric dihydrolipoate and apoferritin, and subsequent identification of the oxidation state of ferritin-bound iron, showed that the first metal atoms were taken up in the ferrous form and that this early step was accompanied by accumulation of ferric iron. Total iron uptake increased with the molar ratio of complex to apoprotein and ranged over 25-40% of the iron being supplied. The amount of ferrous iron found inside the protein did not exceed 50-60 mol iron/mol ferritin after a 48-h incubation. At this time, ferric iron represented a significant fraction of the iron found in the isolated ferritin. Analytical and spectroscopic data indicated that fractional rates and equilibria for disassembly of the ferric complex in the presence of apoferritin were independent of the concentration of the protein and of the complex itself.     

Iron supply to tobacco plants through foliar application of iron citrate and ferric dimerum acid

Experiments to assess the re-greening and the distribution of leaf-applied iron (Fe) within the aerial organs were developed with tobacco ( Nicotiana tabacum L.) plants. Fe (III)-citrate and Fe-dimerum acid were applied to a part of the leaf and plant re-greening was monitored for 6 weeks after treatment. Fluid Fe increments associated with foliar Fe application were measured within 3 days after application. Fe distribution from the site of application within the leaf and the untreated above-ground plant parts was evaluated. Leaf fluid Fe concentration was determined by a novel procedure based on Fe chelation by desferrioxamine E and high performance liquid chromatography separation on a reversed-phase column. The ferrioxamine E method enabled accurate determination of small amounts of Fe present in leaf fluid. Foliar Fe-dimerum acid and Fe (III)-citrate treatment to chlorotic tobacco plants induced new growth re-greening. It was concluded that the applied Fe-containing compounds penetrated the leaf, were distributed within the plant and that Fe supplied as both complexes could be utilized by the plant cell.     

Iron uptake from ferrichrome A and iron citrate in Ustilago sphaerogena.

Double radioactive label transport assays with iron, chromium, and gallium chelates were used to investigate the mechanism of iron uptake by Ustilago sphaerogena. In iron-deficient cells, ferrichrome A iron was taken up without appreciable uptake of the ligand. Iron-sufficient cells partially accumulated the ligand with the metal. The chromium- and gallium-containing analogs of ferrichrome A were transported as intact chelates. Ferrichrome A iron uptake was inhibited by dipyridyl. The data suggest that the intact ferrichrome A chelate binds to a specific receptor, the iron is then separated from the ligand at the membrane by reduction, and the metal is released to the inside of the cell while the ligand is released to the exterior. The reduction step is not transport rate limiting. Iron chelated to citrate was taken up by an energy-dependent process. The citrate ligand was not taken up with the metal. Uptake was sensitive to dipyridyl and ferrozine. Chromic ion chelated to citrate was not transported, suggesting that the iron, rather than the chelate, is recognized by the receptor or that reduction of the metal is required for transport.     

Characterization of autolysins from Mycobacterium smegmatis.

This study demonstrates, for the first time, the autolytic enzymes associated with mycobacterial cell walls. Based on the release of radioactivity and ninhydrin-reactive material from isolated cell walls, it was shown that maximum activity occurs during the late log phase of growth and at a buffer pH of about 8.0. Chemical analyses of autolytic digests of isolated cell walls indicated that at least three autolysins are active under the conditions used. These are N-glycolylmuramic acid-L-alanine amidase, an aminopeptidase that releases L-alanine, and an endopeptidase that solubilizes and L-alanyl-D-glutamic acid dippetide. No other endopeptidase, carboxypeptidase, or glycosidase activity was detected.     

Role of porins in iron uptake by Mycobacterium smegmatis

Many bacteria rely on siderophores to extract iron from the environment. However, acquisition of iron-loaded siderophores is dependent on high-affinity uptake systems that are not produced under high-iron conditions. The fact that bacteria are able to maintain iron homeostasis in the absence of siderophores indicates that alternative iron acquisition systems exist. It has been speculated that such low-affinity uptake of iron in Gram-negative bacteria includes diffusion of iron ions or chelates across the outer membrane through porins. The outer membrane of the saprophytic Mycobacterium smegmatis contains the Msp family of porins, which enable the diffusion of small and hydrophilic solutes, such as monosaccharides, amino acids, and phosphate. However, it is unknown how cations cross the outer membrane of mycobacteria. Here, we show that the Msp porins of M. smegmatis are involved in the acquisition of soluble iron under high-iron conditions. Uptake of ferric ions by a triple porin mutant was reduced compared to wild-type (wt) M. smegmatis. An intracellular iron reporter indicated that derepression of iron-responsive genes occurs at higher iron concentrations in the porin mutant. This was consistent with the finding that the porin mutant produced more siderophores under low-iron conditions than wt M. smegmatis. In contrast, uptake of the exochelin MS, the main siderophore of M. smegmatis, was not affected by the lack of porins, indicating that a specific outer membrane siderophore receptor exists. These results provide, to our knowledge, the first experimental evidence that general porins are indeed the outer membrane conduit of low-affinity iron acquisition systems in bacteria.     

Iron uptake with ferripyochelin and ferric citrate by Pseudomonas aeruginosa.

Pyochelin is an iron-binding compound produced by Pseudomonas aeruginosa and demonstrates siderophore activity by its involvement in iron transport. During the transport process, an energy-independent association of [55Fe]ferripyochelin with bacteria occurred within the initial 30 s of reaction, followed by an energy-dependent accumulation of iron. The energy-independent association with iron appeared to be at the surface of the bacteria because the iron could be washed from the cells with thioglycolate, whereas accumulated iron was not washed from the bacteria. Energy-independent association of iron with bacteria and energy-dependent accumulation of iron in the presence of ferripyochelin varied concomitantly in cells grown under various conditions, but pyochelin synthesis appeared to be controlled separately. 55Fe complexed with citrate was also taken up by P. aeruginosa with a lower level of initial cell association. Bacterial mechanisms for iron uptake from ferric citrate were present in cells grown in a variety of media and were in lowest levels in cells grown in citrate. The synthesis of bacterial components for iron uptake from ferric citrate and from ferripyochelin was inhibited by high concentrations of iron supplied in growth media.     

A genomic view of sugar transport in Mycobacterium smegmatis and Mycobacterium tuberculosis

We present a comprehensive analysis of carbohydrate uptake systems of the soil bacterium Mycobacterium smegmatis and the human pathogen Mycobacterium tuberculosis. Our results show that M. smegmatis has 28 putative carbohydrate transporters. The majority of sugar transport systems (19/28) in M. smegmatis belong to the ATP-binding cassette (ABC) transporter family. In contrast to previous reports, we identified genes encoding all components of the phosphotransferase system (PTS), including permeases for fructose, glucose, and dihydroxyacetone, in M. smegmatis. It is anticipated that the PTS of M. smegmatis plays an important role in the global control of carbon metabolism similar to those of other bacteria. M. smegmatis further possesses one putative glycerol facilitator of the major intrinsic protein family, four sugar permeases of the major facilitator superfamily, one of which was assigned as a glucose transporter, and one galactose permease of the sodium solute superfamily. Our predictions were validated by gene expression, growth, and sugar transport analyses. Strikingly, we detected only five sugar permeases in the slow-growing species M. tuberculosis, two of which occur in M. smegmatis. Genes for a PTS are missing in M. tuberculosis. Our analysis thus brings the diversity of carbohydrate uptake systems of fast- and a slow-growing mycobacteria to light, which reflects the lifestyles of M. smegmatis and M. tuberculosis in their natural habitats, the soil and the human body, respectively.     

Characterization of a porin from Mycobacterium smegmatis.

A pore-forming protein with an Mr of 40,000 has been extracted from the cell wall of Mycobacterium smegmatis with buffer containing the detergent Zwittergent 3-12 and 0.5 M NaCl and purified on an anion-exchange column. Although the pore diameter was large (2 nm), the specific activity was much lower than those of nonspecific porin channels of enteric bacteria. The channel allowed the permeation of small hydrophilic molecules such as sugars and amino acids. Its N-terminal sequence did not show any similarity to those of other porins sequenced so far.     

Transport and utilization of rhizoferrin bound iron in Mycobacterium smegmatis

Transport and metabolization of iron bound to the fungal siderophore rhizoferrin was analyzed by transport kinetics, Mössbauer and EPR spectroscopy. Saturation kinetics (v _max=24.4 pmol/(mg min), K _m=64.4M) and energy dependence excluded diffusion and provided evidence for a rhizoferrin transport system in M. smegmatis. Based on the spectroscopic techniques indications for intracellular presence of the ferric rhizoferrin complex were found. This feature could be of practical importance in the search of novel drugs for the treatment of mycobacterial infections. EPR and Mössbauer spectroscopy revealed different ferritin mineral cores depending on the siderophore iron source. This finding was interpreted in terms of different protein shells, i.e. two types of ferritins.