Effect of iron on the production of pyoverdine and outer membrane proteins ofPseudomonas aeruginosa PA01

Pseudomonas aeruginosa PA01 grown under iron-deficient conditions excreted a yellowish-green pigment, pyoverdine, which has been reported as a siderophore of this bacterium. Addition of iron to the culture medium increased bacterial growth but repressed the production of pyoverdine. Iron levels of the culture medium also had a significant role in the composition of outer membrane proteins ofP. aeruginosa PA01.     

Mössbauer spectroscopy and electron paramagnetic resonance studies of iron metabolites inPseudomonas aeruginosa: Fe2+ and Fe3+ ferritin in57ferripyoverdine incubated cells and57ferric citrate fed cells

Pseudomonas aeruginosa samples were studied using Mössbauer spectroscopy and electron paramagnetic resonance (EPR). Samples included whole cells, membranes, and soluble fractions from cells which had been grown with⁵⁷ferric chloride,⁵⁷ferric citrate or incubated with⁵⁷ferripyoverdine. These experiments show for the first time thatP. aeruginosa can accumulate iron in a bacterioferritin when grown under conditions of iron limitation and incubated with its cognate ferrisiderophore, ferripyoverdine. Soluble fraction fromP. aeruginosa cells which were grown iron starved and incubated with⁵⁷ferripyoverdine for 120 min showed the presence of both a ferric and ferrous complex whose Mössbauer spectra matched that of bacterioferritin extracted fromAzotobacter vinelandii and whose EPR spectra showed a characteristic ferritin-like resonance. A second soluble fraction sample from cells which had been grown with⁵⁷ferric citrate also showed the presence of a species with the same EPR and Mössbauer parameters. In addition Western blotting confirmed the presence of bacterioferritin in the soluble fraction of the cells which had been incubated with ferripyoverdine.     

FERRIC CHELATE REDUCTASE ACTIVITY AS AFFECTED BY THE IRON‐LIMITED GROWTH RATE IN FOUR SPECIES OF UNICELLULAR GREEN ALGAE (CHLOROPHYTA)1

Four species of green algae (Chlorella kessleri Fott et Nováková, Chlorococcum macrostigmatum Starr, Haematococcus lacustris[Girod‐Chantrans] Rostaf., Stichococcus bacillaris Näg.) were grown in iron‐limited chemostats and under phosphate limitation and iron (nutrient) sufficiency. For all four species, steady‐state culture density declined with decreasing degree of iron limitation (increasing iron‐limited growth rate), whereas chl per cell or biovolume increased. Plasma membrane ferric chelate reductase activity was enhanced by iron limitation in all species and suppressed by phosphate limitation and iron sufficiency. These results confirm previous work that C. kessleri uses a reductive mechanism of iron acquisition and also suggest that the other three species use the same mechanism. Although imposition of iron limitation led to enhanced activities of ferric chelate reductase in all species, the relationship between ferric chelate reductase activity and degree of iron limitation varied. Ferric chelate reductase activity in C. macrostigmatum and S. bacillaris was an inverse function of the degree of iron limitation, with the most rapidly growing iron‐limited cells exhibiting the highest ferric chelate reductase activity. In contrast, ferric chelate reductase activity was only weakly affected by the degree of iron limitation in C. kessleri and H. lacustris. Calculation of ferric reductase activity per unit chl allowed a clear differentiation between iron‐limited and iron‐sufficient cells. The possible extension of the ferric chelate reductase assay to investigate the absence or presence of iron limitation in natural waters may be feasible, but it is unlikely that the assay could be used to estimate the degree of iron limitation.     

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.     

Overexpression of AtFRO6 in transgenic tobacco enhances ferric chelate reductase activity in leaves and increases tolerance to iron-deficiency chlorosis

The Arabidopsis gene FRO6(AtFRO6) encodes ferric chelate reductase and highly expressed in green tissues of plants. We have expressed the gene AtFRO6 under the control of a 35S promoter in transgenic tobacco plants. High-level expression of AtFRO6 in transgenic plants was confirmed by northern blot analysis. Ferric reductase activity in leaves of transgenic plants grown under iron-sufficient or iron-deficient conditions is 2.13 and 1.26 fold higher than in control plants respectively. The enhanced ferric reductase activity led to increased concentrations of ferrous iron and chlorophyll, and reduced the iron deficiency chlorosis in the transgenic plants, compared to the control plants. In roots, the concentration of ferrous iron and ferric reductase activity were not significantly different in the transgenic plants compared to the control plants. These results suggest that FRO6 functions as a ferric chelate reductase for iron uptake by leaf cells, and overexpression of AtFRO6 in transgenic plants can reduce iron deficiency chlorosis.     

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.     

Studies on siderophore production and effect of iron deprivation on the outer membrane proteins of Madurella mycetomatis

The purpose of this investigation was to determine whether Madurella mycetomatis, the most frequent agent of eumycotic mycetomas, produces siderophores and synthesizes new outer membrane proteins under iron-starvation conditions. Siderophore production, only of the hydroxamate type, was demonstrated in all nine strains tested. It was regulated by extracellular iron concentrations. Under iron-restricted conditions, M. mycetomatis expressed various outer membrane iron-regulated proteins, particularly of 24-kilodalton, that may participate in iron metabolism.     

Rapid evolution of a bacterial iron acquisition system

Under iron limitation, bacteria scavenge ferric (Fe³⁺) iron bound to siderophores or other chelates from the environment to fulfill their nutritional requirement. In gram‐negative bacteria, the siderophore uptake system prototype consists of an outer membrane transporter, a periplasmic binding protein and a cytoplasmic membrane transporter, each specific for a single ferric siderophore or siderophore family. Here, we show that spontaneous single gain‐of‐function missense mutations in outer membrane transporter genes of Bradyrhizobium japonicum were sufficient to confer on cells the ability to use synthetic or natural iron siderophores, suggesting that selectivity is limited primarily to the outer membrane and can be readily modified. Moreover, growth on natural or synthetic chelators required the cytoplasmic membrane ferrous (Fe²⁺) iron transporter FeoB, suggesting that iron is both dissociated from the chelate and reduced to the ferrous form within the periplasm prior to cytoplasmic entry. The data suggest rapid adaptation to environmental iron by facile mutation of selective outer membrane transporter genes and by non‐selective uptake components that do not require mutation to accommodate new iron sources.     

Responses to iron deficiency in Arabidopsis thaliana: The Turbo iron reductase does not depend on the formation of root hairs and transfer cells

Arabidopsis thaliana (L.) Heynh. Columbia wild type and a root hair-less mutant RM57 were grown on iron-containing and iron-deficient nutrient solutions. In both genotypes, ferric chelate reductase (FCR) of intact roots was induced upon iron deficiency and followed a Michaelis-Menten kinetic with a K _m of 45 and 54 M Fe^III-EDTA and a V _max of 42 and 33 nmol Fe²⁺·(g FW)^–1·min^–1 for the wild type and the mutant, respectively. The pH optimum for the reaction was around pH 5.5. The approximately four fold stimulation of FCR activity was independent of formation of root hairs and/or transfer cells induced by iron deficiency. Iron-deficiency-induced chlorosis and the development of a rigid root habit disappeared when ferric chelate was applied to the leaves, while FCR activity remained unchanged. The time course of the responses to iron deficiency showed that morphological and physiological responses were controlled separately.Abbreviations FCR ferric chelate reductase - FW fresh weight Thanks are due to Klaas Sjollema (Department of Electronmicroscopy, University of Groningen, The Netherlands) for help with the electron microscopy sample preparation and especially to Dr. Uwe Santore (Heinrich-Heine-University for electron microscopy. This work was supported by the SCIENCE programm of the European community; P.R.M.) and a Personal Research Grant by the Ministerium für Wissenschaft und Forschung of Nordrhein-Westfalen (P.R.M.) and last, not least by the productive discussions in ECOTRANS B.V.     

<Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> phage endolysin can enhance permeability of<Emphasis Type="Italic"> Pseudomonas aeruginosa</Emphasis> outer membrane and induce cell lysis

To determine the function of the C-terminal region of Bacillus amyloliquefaciens phage endolysin on Pseudomonas aeruginosa lysis, the permeabilization of the outer membrane of P. aeruginosa was analyzed. Glu-15 to His (E15H) and Thr-32 to Glu (T32E) substitutions were introduced into the Bacillus phage endolysin. Neither E15H nor T32E substitution induced enzymatic and antibacterial activities. These two, Glu-15 and Thr-32, were considered to be the active center of the enzyme. The addition of purified E15H and T32E proteins to P. aeruginosa cells induced the release of periplasmic -lactamase from the cells, indicating that both proteins enhance permeabilization of the outer membrane. However, the addition of E15H and T32E proteins to P. aeruginosa cells did not induce the release of cytoplasmic ATP from the cells. These results indicate that the antibacterial activity of the endolysin requires both the C-terminal enhancement of the permeabilization of the P. aeruginosa outer membrane and N-terminal enzymatic activity.     

Utilization of Iron Sources and Its Possible Roles in the Pathogenesis of Vibrio parahaemolyticus

Vibrio parahaemolyticus is an important enteropathogen in Japan, Taiwan and other coastal regions. The influence of the regulation of iron on the pathogenesis of this pathogen has not been well characterized. The growth of pathogenic and non-pathogenic strains of V. parahaemolyticus on iron-limited agar plates was stimulated by ferritin, lactoferrin and transferrin at 30 μM , and also by hemin, hemoglobin and ferric ammonium citrate at 100 μM . Spontaneous iron-utilizing mutant strains (mutants) were derived from a clinical strain, ST550. Compared with the parent strain, lowered virulence was demonstrated for these mutants, as assayed by adult mouse and suckling mouse models. The in vivo growth and enterotoxigenicity of these mutants were also lower in the suckling mice. Adherence of the mutants to excised mouse intestine was lower as demonstrated by scanning electron microscopy. The iron-regulated outer membrane protein profile also changed in selected mutants. These results indicate that iron-regulated outer membrane proteins and other unknown factors associated with iron utilization may have profound influences, besides iron acquisition, on the pathogenesis of V. parahaemolyticus.     

The Bradyrhizobium japonicum fsrB gene is essential for utilization of structurally diverse ferric siderophores to fulfill its nutritional iron requirement

In Bradyrhizobium japonicum, iron uptake from ferric siderophores involves selective outer membrane proteins and non-selective periplasmic and cytoplasmic membrane components that accommodate numerous structurally diverse siderophores. Free iron traverses the cytoplasmic membrane through the ferrous (Fe²⁺) transporter system FeoAB, but the other non-selective components have not been described. Here, we identify fsrB as an iron-regulated gene required for growth on iron chelates of catecholate- and hydroxymate-type siderophores, but not on inorganic iron. Utilization of the non-physiological iron chelator EDDHA as an iron source was also dependent on fsrB. Uptake activities of ⁵⁵Fe³⁺ bound to ferrioxamine B, ferrichrome or enterobactin were severely diminished in the fsrB mutant compared with the wild type. Growth of the fsrB or feoB strains on ferrichrome were rescued with plasmid-borne E. coli fhuCDB ferrichrome transport genes, suggesting that FsrB activity occurs in the periplasm rather than the cytoplasm. Whole cells of an fsrB mutant are defective in ferric reductase activity. Both whole cells and spheroplasts catalyzed the demetallation of ferric siderophores that were defective in an fsrB mutant. Collectively, the data support a model whereby FsrB is required for reduction of iron and its dissociation from the siderophore in the periplasm, followed by transport of the ferrous ion into the cytoplasm by FeoAB.     

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.     

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.     

Influence of growth rate and iron limitation on the expression of outer membrane proteins and enterobactin by Klebsiella pneumoniae grown in continuous culture.

The influence of the growth rate on outer membrane protein composition and enterobactin production was studied with Klebsiella pneumoniae grown under conditions of iron limitation in chemostats. More enterobactin was produced at fast (D = 0.4 h-1) and slow (D = 0.1 h-1) growth rates in continuous cultures than in either logarithmic- or stationary-phase batch cultures. When the growth rate was controlled under conditions of carbon limitation and the iron level was reduced to 0.5 microM, the iron-regulated outer membrane proteins and enterobactin were induced at the fast growth rate. At the slow growth rate, although the iron-regulated outer membrane proteins were barely visible, a significant level of enterobactin was still produced. These results suggest that under conditions of either carbon or iron limitation, the growth rate can influence the induction of the high-affinity iron uptake system of K. pneumoniae. Other outer membrane proteins, including a 39-kilodalton peptidoglycan-associated protein, were found to vary with the growth rate and nutrient limitation.     

A role for PchHI as the ABC transporter in iron acquisition by the siderophore pyochelin in Pseudomonas aeruginosa

Iron is an essential nutrient for bacterial growth but poorly bioavailable. Bacteria scavenge ferric iron by synthesizing and secreting siderophores, small compounds with a high affinity for iron. Pyochelin (PCH) is one of the two siderophores produced by the opportunistic pathogen Pseudomonas aeruginosa. After capturing a ferric iron molecule, PCH-Fe is imported back into bacteria first by the outer membrane transporter FptA and then by the inner membrane permease FptX. Here, using molecular biology, ⁵⁵Fe uptake assays, and LC–MS/MS quantification, we first find a role for PchHI as the heterodimeric ABC transporter involved in the siderophore-free iron uptake into the bacterial cytoplasm. We also provide the first evidence that PCH is able to reach the bacterial periplasm and cytoplasm when both FptA and FptX are expressed. Finally, we detected an interaction between PchH and FptX, linking the ABC transporter PchHI with the inner permease FptX in the PCH-Fe uptake pathway. These results pave the way for a better understanding of the PCH siderophore pathway, giving future directions to tackle P. aeruginosa infections.     

Susceptibility of Pseudomonas aeruginosa Urinary Tract Isolates and Influence of Urinary Tract Conditions on Antibiotic Tolerance

Pseudomonas aeruginosa is an opportunistic human pathogen, which can cause severe urinary tract infections (UTIs). Because of the high intrinsic antibiotic resistance of P. aeruginosa and its ability to develop new resistances during antibiotic treatment, these infections are difficult to eradicate. The antibiotic susceptibility of 32 P. aeruginosa isolates from acute and chronic UTIs were analysed under standardized conditions showing 19% multi-drug resistant strains. Furthermore, the antibiotic tolerance of two P. aeruginosa strains to ciprofloxacin and tobramycin was analysed under urinary tract-relevant conditions which considered nutrient composition, biofilm growth, growth phase, and oxygen concentration. These conditions significantly enhance the antibiotic tolerance of P. aeruginosa up to 6000-fold indicating an adaptation of the bacterium to the specific conditions present in the urinary tract. This reversible phenomenon is possibly due to the increased formation of persister cells and is based on iron limitation in artificial urine. The results suggest that the general high antibiotic resistance of P. aeruginosa urinary tract isolates together with the increasing tolerance of P. aeruginosa grown under urinary tract conditions decrease the efficiency of antibiotic treatment of UTIs.     

Expression profiling of the Arabidopsis ferric chelate reductase (FRO) gene family reveals differential regulation by iron and copper

The Arabidopsis FRO2 gene encodes the iron deficiency-inducible ferric chelate reductase responsible for reduction of iron at the root surface; subsequent transport of iron across the plasma membrane is carried out by a ferrous iron transporter (IRT1). Genome annotation has identified seven additional FRO family members in the Arabidopsis genome. We used real-time RT-PCR to examine the expression of each FRO gene in different tissues and in response to iron and copper limitation. FRO2 and FRO5 are primarily expressed in roots while FRO8 is primarily expressed in shoots. FRO6 and FRO7 show high expression in all the green parts of the plant. FRO3 is expressed at high levels in roots and shoots, and expression of FRO3 is elevated in roots and shoots of iron-deficient plants. Interestingly, when plants are Cu-limited, the expression of FRO6 in shoot tissues is reduced. Expression of FRO3 is induced in roots and shoots by Cu-limitation. While it is known that FRO2 is expressed at high levels in the outer layers of iron-deficient roots, histochemical staining of FRO3-GUS plants revealed that FRO3 is predominantly expressed in the vascular cylinder of roots. Together our results suggest that FRO family members function in metal ion homeostasis in a variety of locations in the plant.     

Studies on iron uptake and its effect on the outer membrane proteins ofPseudomonas stuzeri RC-7

Pseudomonas stutzeri RC-7, grown under iron-deficient conditions, produced catecholtype siderophore. Iron uptake was enhanced by the presence of its own siderophore. The production of iron-binding protein with molecular weight of 55,000 in the outer membrane was found to be suppressed in iron-supplemented conditions. In contrast, another protein with molecular weight of 42,000 was induced under the same conditions.     

Influence of Iron Deprivation on the Membrane Composition of Anacystis nidulans

Cultures of the cyanobacterium Anacystis nidulans were grown under iron-deficient conditions and then restored by the addition of iron. Membrane proteins from iron-deficient and iron-restored cells were analyzed by lithium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The incorporation of [³⁵S]sulfate into membrane proteins and lactoperoxidase-catalyzed ¹²⁵I iodination were used to monitor the rates of polypeptide biosynthesis and surface exposure of membrane proteins, respectively. These polypeptide profiles revealed major differences in the membrane composition of iron-deficient and normal cells. Iron deficiency caused a decrease in the amount of certain important membrane proteins, reflecting a decreased rate of biosynthesis of these peptides. Several photosystem II peptides also showed an increase in surface exposure after iron stress. In addition, iron deficiency led to the synthesis of proteins at 34 and 52 kilodaltons which were not present in normal cells. When iron was restored to a deficient culture, a metabolic sequence was initiated within the first 12 h after the addition of iron which led to phenotypically normal cells. Pulse labeling with [³⁵S]sulfate during this period demonstrated that iron addition initiates a coordinated pattern of synthesis that leads to the assembly of normal membranes.