High-iron Medium Research Articles

Physiological and molecular genetic evaluation of the dechlorination agent, pyridine-2,6-bis(monothiocarboxylic acid) (PDTC) as a secondary siderophore of Pseudomonas.

The bacterial metabolite and transition metal chelator pyridine-2,6-dithiocarboxylic acid (PDTC), promotes a novel and effective means of dechlorination of the toxic and carcinogenic pollutant, carbon tetrachloride. Pyridine-2,6-dithiocarboxylic acid has been presumed to act as a siderophore in the Pseudomonas strains known to produce it. To explore further the physiological function of PDTC production, we have examined its regulation, the phenotype of PDTC-negative (pdt) mutants, and envelope proteins associated with PDTC in P. putida strain DSM 3601. Aspects of the regulation of PDTC production and outer membrane protein composition were consistent with siderophore function. Pyridine-2,6-dithiocarboxylic acid production was coordinated with production of the well-characterized siderophore pyoverdine; exogenously added pyoverdine led to decreased PDTC production, and added PDTC led to decreased pyoverdine production. Positive regulation of a chromosomal pdtI-xylE transcriptional fusion, and of a 66 kDa outer membrane protein (IROMP), was seen in response to exogenous PDTC. Tests with transition metal chelators indicated that PDTC could provide a benefit under conditions of metal limitation; the loss of PDTC biosynthetic capacity caused by a pdtI transposon insertion resulted in increased sensitivity to 1,10-phenanthroline, a chelator that has high affinity for a range of divalent transition metals (e.g. Fe(2+), Cu(2+), Zn(2+)). Exogenously added PDTC could also suppress a phenotype of pyoverdine-negative (Pvd-) mutants, that of sensitivity to EDDHA, a chelator with higher affinity and specificity for Fe(3+). Measurement of 59Fe incorporation showed uptake from 59Fe:PDTC by DSM 3601 grown in low-iron medium, but not by cells grown in high iron medium, or by the pdtI mutant, which did not show expression of the 66 kDa envelope protein. These data verified a siderophore function for PDTC, and have implicated it in the uptake of transition metals in addition to iron.

Mitochondrial functional interactions between frataxin and Isu1p, the iron–sulfur cluster scaffold protein, in Saccharomyces cerevisiae

Friedreich’s ataxia is caused by a deficit in the mitochondrial protein frataxin. The present work demonstrates that in vivo yeast frataxin Yfh1p and Isu1p, the mitochondrial scaffold protein for the Fe–S cluster assembly, have tightly linked biological functions, acting in concert to promote the Fe–S cluster assembly. A synthetic lethal screen on high iron media with the mild G107D yfh1 mutant has specifically identified Isu1p. Analysis of the cellular phenotypes resulting from pairwise combinations of yfh1 and isu1 mutations, and cross-linking experiments in isolated mitochondria provide evidence for a direct interaction between Yfh1p and Isu1p.

A Dominant Allele of PDR1 Alters Transition Metal Resistance in Yeast

A yeast mutant was found to have defective growth on low iron medium despite a normal high affinity iron transport system. The phenotype results from a gain of function mutation in PDR1, which encodes a transcription factor that acts as a regulator of pleiotropic drug resistance in Saccharomyces cerevisiae. The mutant allele, PDR1(R821H), was found to result in increased expression of at least 19 genes, three of which are ATP-binding cassette (ABC) transporters. Expression of at least six genes was required to show the low iron growth defect. Wild type cells transformed with the PDR1(R821H) allele or a PDR1 dominant allele (PDR1-3) showed the low iron growth defect as well as increased resistance to drugs such as cycloheximide and oligomycin. Transformation of PDR1(R821H) into Deltaccc1 cells, which were previously shown to have increased sensitivity to high iron medium because of defective vacuolar iron storage (Li, L., Chen, O. S., Ward, D. M., and Kaplan, J. (2001) J. Biol. Chem. 276, 29515-29519), conferred resistance to high iron medium. Cells expressing PDR1(R821H) also showed increased resistance to copper and manganese because of increased metal export. These results suggest that expression of PDR1-regulated genes affects both efflux and storage of transition metals.

Identification and genetic characterization of PmrA-regulated genes and genes involved in polymyxin B resistance in Salmonella enterica serovar typhimurium.

Salmonella enterica serovar Typhimurium encounters antimicrobial peptides (AP) within the phagosomes of professional phagocytes and at intestinal mucosal surfaces. Salmonella serovar Typhimurium utilizes the two-component regulatory system PmrA-PmrB, which is activated in response to the environmental conditions encountered in vivo, to regulate resistance to several AP, including polymyxin B (PM). Random MudJ transposon mutagenesis was used to identify PmrA-PmrB-regulated genes, as well as genetic loci necessary for PM resistance. Three different phenotypic classes of genes were identified: those necessary for PM resistance and regulated by PmrA, those necessary for PM resistance and not regulated by PmrA, and PmrA-regulated genes not required for PM resistance. Loci identified as necessary for PM resistance showed between 6- and 192-fold increased sensitivities to PM, and transposon insertion sites include surA, tolB, and gnd. PmrA-regulated loci identified included dgoA and yibD and demonstrated 500- and 2,500-fold activation by PmrA, respectively. The role of the identified loci in aminoarabinose modification of lipid A was determined by paper chromatography. The gnd mutant demonstrated a loss of aminoarabinose from lipid A, which was suggested to be due to a polar effect on the downstream gene pmrE. The remaining PM(s) mutants (surA and tolB), as well as the two PmrA-regulated gene (yibD and dgoA) mutants, retained aminoarabinose on lipid A. yibD, dgoA, and gnd (likely affecting pmrE) played no role in PmrA-regulated resistance to high iron concentrations, while surA and tolB mutations grew poorly on high iron media. All PM(s) mutants identified in this study demonstrated a defect in virulence compared to wild-type Salmonella serovar Typhimurium when administered orally to mice, while the PmrA-regulated gene (yibD and dgoA) mutants showed normal virulence in mice. These data broaden our understanding of in vivo gene regulation, lipopolysaccharide modification, and mechanisms of resistance to AP in enteric bacteria.

Deletion of the Mitochondrial Carrier Genes MRS3 andMRS4 Suppresses Mitochondrial Iron Accumulation in a Yeast Frataxin-deficient Strain

The mitochondrial solute carriers Mrs3p and Mrs4p were originally isolated as multicopy suppressors of intron splicing defects. We show here that MRS4 is co-regulated with the iron regulon genes, and up-regulated in a strain deficient for Yfh1p, the yeast homologue of human frataxin. Using in vivo 55Fe cell radiolabeling we show that in glucose-grown cells mitochondrial iron accumulation is 5-15 times higher in deltaYFH1 than in wild-type strain. However, although in a deltaYFH1deltaMRS3deltaMRS4 strain, the intracellular 55Fe content is extremely high, the mitochondrial iron concentration is decreased to almost wild-type levels. Moreover, deltaYFH1deltaMRS3deltaMRS4 cells grown in high iron media do not lose their mitochondrial genome. Conversely, a deltaYFH1 strain overexpressing MRS4 has an increased mitochondrial iron content and no mitochondrial genome. Therefore, MRS4 is required for mitochondrial iron accumulation in deltaYFH1 cells. Expression of the iron regulon and intracellular 55Fe content are higher in a deltaMRS3deltaMRS4 strain than in the wild type. Nevertheless, the mitochondrial 55Fe content, a balance between iron uptake and exit, is decreased by a factor of two. Moreover, 55Fe incorporation into heme by ferrochelatase is increased in an MRS4-overexpressing strain. The function of MRS4 in iron import into mitochondria is discussed.

Regulation of ornibactin biosynthesis and N-acyl-L-homoserine lactone production by CepR in Burkholderia cepacia.

The CepR-CepI quorum-sensing system has been shown to regulate production of the siderophore ornibactin, extracellular proteases, and N-octanoyl-homoserine-L-lactone (OHL) in Burkholderia cepacia strain K56-2. To examine the effect of cepIR on production of other siderophores, cepR mutants were constructed in strains that produce pyochelin in addition to salicylic acid and ornibactins. Pc715j-R1 (cepR::tp) hyperproduced ornibactin but produced parental levels of pyochelin and salicylic acid, suggesting that CepR is a negative regulator of ornibactin synthesis but not pyochelin or salicylic acid. Pc715j-R1 was also protease deficient and OHL negative. The effects of cepR on ornibactin biosynthetic genes were examined by constructing cepR pvdA-lacZ and cepR pvdD-lacZ mutants and monitoring beta-galactosidase activity. There was an increase in expression of pvdA in the cepR mutant compared to the level in its parent strain in both low- and high-iron media during stationary phase. When the outer membrane protein profiles of a cepR mutant and the wild-type strain were compared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, there did not appear to be any difference in levels of expression of the ornibactin receptor. Experiments with cepI-lacZ and cepR-lacZ transcriptional fusions indicated that cepI was not expressed in the cepR mutant and that cepR acts as a negative regulator of its own expression. By a thin-layer chromatography assay for N-acyl homoserine lactones, OHL and N-hexanoyl-L-homoserine lactone (HHL) were detectable in K56-2 and Pc715j, both wild-type strains. OHL was not detectable and HHL was only weakly detectable in the cepI and cepR mutants. These results suggest that CepR is both a positive and negative transcriptional regulator and that CepR may influence the expression of ornibactin biosynthetic genes in addition to the expression of the cepIR quorum-sensing system.

Morphological and Physiological Differences in Synechococcus elongatus during Continuous Cultivation at High Iron, Low Iron, and Iron Deficient Medium

Thermophilic unicellular cyanobacterium Synechococcus elongatus Nag. var. thermalis Geitl. strain Kovrov 1972/8 was cultivated in continuous flow reactor to simulate conditions occurring in nature in regions with low iron concentration. Two degrees of iron deprivation were established: (a) low iron (LI) conditions (9.0 µM Fe) when cells still maintained maximal growth rate but already exhibited changes in photosynthetic apparatus, and (b) iron deficient (ID) conditions (0.9 µM Fe) when cell growth rate decreased and extensive morphological and functional changes were observed. A decrease in the cellular content of phycobilin antenna was observed in both ID and LI cells and an increase of carotenoid concentration only in the ID culture. Morphologically, ID cells showed a decrease in the amount of phycobilins and in the number of thylakoid membranes. This suggests that S. elongatus responds to decrease in iron availability by substitution of the phycobilisomes by antennae containing chlorophyll (Chl) and carotenoids. Photochemical activity of photosystem (PS) 2, determined as Fv/Fm ratio was similar in high iron (HI) and LI cultures and approximately five times lower in ID culture. On the other hand, the activity of the whole electron transport chain showed the opposite tendency: the relative rates of the CO2-dependent oxygen evolution in HI : LI : ID cultures were approximately 1 : 2 : 4. Thus in nutrient stress the photosynthetic apparatus preserved its activity despite the decrease in the amount of both Chl-binding complexes and thylakoid membranes.

Iron modulates phenotypic variation and phosphorylation of P270 in double-stranded RNA virus-infected Trichomonas vaginalis.

Trichomonas vaginalis infected with a double-stranded RNA virus undergoes phenotypic variation on the basis of surface versus cytoplasmic expression of the immunogenic protein P270. Examination of batch cultures by flow cytofluorometry with monoclonal antibody (MAb) to P270 yields both fluorescent and nonfluorescent trichomonads. Greater numbers and intensity of fluorescent organisms with surface P270 reactive with MAb were evident in parasites grown in medium depleted of iron. Placement of iron-limited organisms in medium supplemented with iron gave increased numbers of nonfluorescent trichomonads. Purified subpopulations of trichomonads with and without surface P270 obtained by fluorescence-activated cell sorting reverted to nonfluorescent and fluorescent phenotypes when placed in high- and low-iron media, respectively. No similar regulation by iron of P270 was evident among virus-negative T. vaginalis isolates or virus-negative progeny trichomonads derived from virus-infected isolates. Equal amounts of P270 were detectable by MAb on immunoblots of total proteins from identical numbers of parasites grown in low- and high-iron media. Finally, P270 was found to be highly phosphorylated in high-iron parasites. Iron, therefore, plays a role in modulating surface localization of P270 in virus-harboring parasites.

Anaerobiosis of Pathogenic Pseudomonas aeruginosa: Suppression of Tox A Gene

Pseudomonas aeruginosa PAO1, and PA 103 strains were allured in TSBD medium in the presence or absence of oxygen with low or high iron medium Both the strains showed higher cell densities with high iron medium under aerobic and anaerobic conditions suggesting that iron is a very important factor for bacterial cell growth. The exotoxin-A assay showed that there was 45% and 79.5% reduction in the toxin yields of PAO, and PA 103 strains respectively when cultured in iron restricted TSBD medium under anaerobic condition, indicating the suppression of Tox A gene, a situation comparable to Cystic Fibrosis (CF) lung infections of pseudomonas.

Iron-responsive gene regulation in a campylobacter jejuni fur mutant.

The expression of iron-regulated systems in gram-negative bacteria is generally controlled by the Fur protein, which represses the transcription of iron-regulated promoters by using Fe2+ as a cofactor. Mutational analysis of the Campylobacter jejuni fur gene was carried out by generation of a set of mutant copies of fur which had a kanamycin or chloramphenicol resistance gene introduced into the regions encoding the N and C termini of the Fur protein. The mutated genes were recombined into the C. jejuni NCTC 11168 chromosome, and putative mutants were confirmed by Southern hybridization. C. jejuni mutants were obtained only when the resistance genes were transcribed in the same orientation as the fur gene. The C. jejuni fur mutant grew slower than the parental strain. Comparison of protein profiles of fractionated C. jejuni cells grown in low- or high-iron medium indicated derepressed expression of three iron-regulated outer membrane proteins with molecular masses of 70, 75, and 80 kDa. Characterization by N-terminal amino acid sequencing showed the 75-kDa protein to be identical to CfrA, a Campylobacter coli siderophore receptor homologue, whereas the 70-kDa protein was identified as a new siderophore receptor homologue. Periplasmic fractions contained four derepressed proteins with molecular masses of 19, 29, 32, and 36 kDa. The 19-kDa protein has been previously identified, but its function is unknown. The cytoplasmic fraction contained two iron-repressed and two iron-induced proteins with molecular masses of 26, 55, 31, and 40 kDa, respectively. The two iron-repressed proteins have been previously identified as the oxidative stress defense proteins catalase (KatA) and alkyl hydroperoxide reductase (AhpC). AhpC and KatA were still iron regulated in the fur mutant, suggesting the presence of Fur-independent iron regulation. Further analysis of the C. jejuni iron and Fur regulons by using two-dimensional gel electrophoresis demonstrated the total number of iron- and Fur-regulated proteins to be lower than for other bacterial pathogens.

Effects of iron and temperature on expression of the Pseudomonas aeruginosa tolQRA genes: role of the ferric uptake regulator.

The tolQRA genes have been recently identified in Pseudomonas aeruginosa PAO. In this study, we examined the effect of iron and temperature on tolQRA expression. A promoterless lacZ gene was introduced downstream of plasmid-encoded tolQ and tolA, and expression was monitored by measuring beta-galactosidase activity of cultures. Addition of 25 microM FeCl3 to the culture medium reduced tolQRA expression by 50 to 60% in PAO but by only 25% in the fur mutant PAO A4. Northern hybridization analysis revealed that iron regulation occurs at the level of transcription and involves the P. aeruginosa ferric uptake regulator (Fur). Primer extension analysis was used to identify the proposed transcriptional start site of tolA. Although a putative Fur box was identified 20 bp upstream of the proposed start site, purified Fur did not bind to the tolA or tolQR promoter regions in an in vitro gel retardation assay. Therefore, iron regulation of the tol genes appears to involve an intermediate regulatory gene. Expression of tolQR and tolA was optimal at 37 degrees C and was reduced by 40 to 50% when cultures were grown at either 42 or 25 degreesC. Growth in high-iron medium at 25 degrees C further reduced tolQR and tolA expression.

Effects of high irradiance and iron concentration on pigment and fatty acid composition in the cyanobacterium Microcystis aeruginosa

Microcystis aeruginosa Kuetzing emend. was exposed to sunlight and artificial illumination at low (1 µM) and high (31 µM) iron concentrations over a 9 h period. With sunlight and low iron, cellular carotenoid and chlorophyll a concentrations decreased linearly with time, and membrane polyunsaturated fatty acids were reduced after 9 h. With sunlight and high iron, there was near-complete loss of all pigments and reduced concentrations of all major fatty acids within 3 h, presumably due to photooxidation; almost all phytol, poly- and monounsaturated fatty acids were absent after 9 h exposure in the high iron medium, indicating total destruction of membranes by iron-enhanced photooxidation. Although caution is required in extrapolating results from laboratory grown cultures to field populations, it is conceivable that high irradiance can influence the composition of cyanobacterial membranes during natural surface blooms. The subsequent survival and regeneration of cyanobacterial cells may be affected under the high iron concentrations which can occur in Australian inland lakes and rivers.

Characterization of lipoprotein IRP1 from Corynebacterium diphtheriae, which is regulated by the diphtheria toxin repressor (DtxR) and iron.

The Corynebacterium diphtheriae irp1 gene is negatively regulated by DtxR and iron. The nucleotide sequence of irp1 revealed that it has homology with genes involved in iron acquisition. Expression of the irp1 gene showed that it encodes a lipoprotein (IRP1) with a predicted size of 38 kDa. Northern blot experiments indicated that transcription from the irp1 promoter is repressed in high-iron medium and suggested that irp1 is part of an iron-regulated operon.

Transcription of the Corynebacterium diphtheriae hmuO gene is regulated by iron and heme.

The hmuO gene is required for the utilization of heme and hemoglobin as iron sources by Corynebacterium diphtheriae. The product of hmuO has homology to eukaryotic heme oxygenases which are involved in the degradation of heme and the release of iron. To investigate the mechanism of hmuO regulation, a promoterless lacZ gene present on the promoter-probe vector pCM502 was placed under transcriptional control of the hmuO promoter. In C. diphtheriae C7, optimal expression from the hmuO promoter was obtained only in the presence of heme or hemoglobin under low-iron conditions. Expression of hmuO in high-iron medium containing heme was repressed five- to sixfold from that seen under low-iron conditions in the presence of heme. Transcription from the hmuO promoter in the absence of heme or hemoglobin was fully repressed in high-iron medium and was expressed at very low levels in iron-depleted conditions. Expression studies with tile hmuO-lacZ fusion construct in C7hm723, a dtxR mutant of C7, and in a hmuO mutant of C. diphtheriae HC1 provided further evidence that transcription of the hmuO promoter is repressed by DtxR and iron and activated by heme. In Escherichia coli, the hmuO promoter was expressed at very low levels under all conditions examined. Gel mobility shift assays and DNase I footprinting experiments indicated that DtxR binds in a metal-dependent manner to a sequence that overlaps the putative hmuO promoter. Total cellular RNA isolated from C. diphtheriae was used to identify the transcriptional start site for the hmuO gene. Northern blot analysis suggested that the hmuO mRNA was monocistronic and that transcription was heme inducible.

Transferrin-iron and proinflammatory cytokines influence iron status and apical iron transport efficiency of Caco-2 intestinal cell line

Endogenous factors that regulate the absorption of dietary iron remain unknown. Differentiated cultures of Caco-2 human intestinal cells grown on membrane inserts were used to study the characteristics of transferrin-iron uptake across the basolateral surface, the effects of transferrin-iron uptake on cellular ferritin content, the transport of apical iron across the monolayer, and the influence of proinflammatory cytokines on these processes. Caco-2 cells accumulated transferrin-iron from the basolateral chamber by a temperature-dependent, saturable process that was enhanced in less differentiated cultures and attenuated by pre-exposure to high-iron medium. Exposure of Caco-2 cells to 10 μmol/L diferric transferrin for 36 hr increased cellular ferritin protein 3.4-fold and decreased the transport of apical 59Fe to the basolateral compartment by 45%. Pretreatment of cells with a combination of interleukin-1β, interleukin-6, and tumor necrosis factor-α increased transferrin-iron uptake by 70% and cellular ferritin content by 54%. Also, cytokine treatment decreased apical iron transport across the monolayer by 40% without altering paracellular transport of mannitol. These results suggest that transferrin-iron and proinflammatory cytokines are capable of modulating the iron status and iron transport activity of intestinal epithelial cells.

Exotoxin A production in Pseudomonas aeruginosa requires the iron-regulated pvdS gene encoding an alternative sigma factor.

Exotoxin A (ETA) is secreted by Pseudomonas aeruginosa under iron-limiting growth conditions. The ETA structural gene, toxA, is regulated at the transcriptional level by the gene products of the regAB operon. The expression of both toxA and regAB is repressed under iron-replete conditions, suggesting a role for the ferric uptake regulator (Fur) in regulation of ETA synthesis; however, the Fur protein does not interact directly with the toxA or the regAB promoters. Evidence is presented that the iron control of ETA synthesis is mediated by a Fur-regulated alternative sigma factor, PvdS, which had initially been identified as a positive activator for the production of the siderophore pyoverdin. In a delta pvdS deletion mutant, ETA was produced at low levels of less than 5% compared to wild type, but still in response to iron starvation, and introduction of a functional pvdS gene on a plasmid fully restored wild-type levels and normal iron regulation of ETA synthesis. Therefore, a functional pvdS locus is essential for ETA production. Neither toxA nor regAB mRNA was detectable in a delta pvdS mutant. Overexpression of pvdS from the tac promoter on a plasmid resulted in a high-level and iron-independent production of ETA in wild-type PAO1, in the delta pvdS strain, but not in a delta regA strain as a host. These findings suggest that PvdS is required for the activation of the regAB promoters. The transcription of regAB and toxA after induction of the P tac-pvdS gene was monitored in cells grown in high-iron medium. While both regAB and toxA were highly expressed during all growth phases under microaerobic conditions, toxA transcripts were detected only during the exponential but not the early stationary phase of growth under aerobic conditions. These results suggest that a second regulatory mechanism besides the Fur-PvdS system is involved in iron regulation of ETA production.

Candida albicans has a cell-associated ferric-reductase activity which is regulated in response to levels of iron and copper.

For survival, pathogenic organisms such as Candida albicans must possess an efficient mechanism for acquiring iron in the iron-restricted environment of the human body. C. albicans can use iron from a variety of sources found within the host. However, it is not clear how biologically active ferrous iron is obtained from these sources. One strategy adopted by some organisms is to reduce iron extracellularly and then specifically transport the ferrous iron into the cell. We have shown that clinical isolates of C. albicans do have a cell-associated ferric-reductase activity. The determination of ferric-reductase activity of cells growing exponentially in either low- or high-iron media over a period of time indicated that C. albicans reductase activity is induced when in low-iron conditions. Moreover, we have demonstrated that C. albicans reductase activity is also regulated in response to the growth phase of the culture, with induction occurring upon exit from stationary phase and maximal levels being reached in early exponential stage irrespective of the iron content of the medium. These results suggest that C. albicans reductase activity is regulated in a very similar manner to the Saccharomyces cerevisiae ferric-reductase. Iron reduction and uptake in S. cerevisiae are closely connected to copper reduction, and possibly copper uptake. In this report we show that iron and copper reduction also appear to be linked in C. albicans. The ferric-reductase activity is negatively regulated by copper. Moreover, quantitative cupric-reductase assays indicated that C. albicans is capable of reducing copper and that this cupric-reductase activity is negatively regulated by both iron and copper. This is the first report that C. albicans has an iron- and copper-mediated ferri-reductase activity.

Cloning and molecular characterization of two genes encoding adhesion proteins involved in Trichomonas vaginalis cytoadherence.

Cytoadherence to the vaginal epithelium is a critical step in infection by the eukaryotic flagellate Trichomonas vaginalis. Four trichomonad surface proteins (AP65, AP51, AP33 and AP23) mediate cytoadherence. The cDNA encoding the AP65 adhesin was isolated from a phagemid cDNA expression library by screening with antiserum and monoclonal antibody (mAb) raised against the purified trichomonad AP65 protein. Two clones, F11.2 and F11.5, coded for immuno-crossreactive recombinant proteins that possessed functional properties equal to the T. vaginalis AP65 adhesin. Analysis of full-length sequences corresponding to the F11.2 and F11.5 cDNAs revealed that both contained 1701-base open reading frames (ORFs) that encoded proteins of 63 281 daltons and 83 087 daltons, respectively. Comparison of the full-length sequences showed 87% identity at the nucleotide level and 91% identity at the protein level. Restriction-enzyme mapping and Southern analysis reaffirmed the distinctness of the F11.2 and F11.5 cDNAs, indicating that two different AP65 genes (now called ap65-1 and ap65-2) are present in the T. vaginalis genome in at least two copies each. Northern analysis detected high levels of transcript of approximately 1.8 kb for both ap65-1 and ap65-2 genes in trichomonads grown only in high-iron medium, confirming the transcriptional regulation of adhesin synthesis by iron. Homology searches revealed significant similarity (38% amino acid identity and 54% nucleotide identity) to malic enzymes. However, purified malic enzyme and mAb to AP65 crossreactive with malic enzyme neither inhibited cytoadherence of T. vaginalis to host cells nor prevented binding of the trichomonad AP65 to HeLa cells in a ligand assay.

Iron regulatory factor expressed from recombinant baculovirus: conversion between the RNA-binding apoprotein and Fe-S cluster containing aconitase.

Iron regulatory factor (IRF) is a cytoplasmic mRNA-binding protein that coordinates post-transcriptionally the expression of several important proteins in iron metabolism. Binding of IRF to iron-responsive elements (IRE) in the 5' untranslated region (UTR) of ferritin and erythroid 5-aminolevulinic acid-synthase mRNAs inhibits their translation, whereas binding to IREs in the 3' UTR of transferrin receptor (TfR) mRNA prevents the degradation of this mRNA. IRF binds RNA strongly after iron deprivation, but is inactive, yet present, under conditions of high cellular iron supply. Recently, IRF was also shown to have aconitase activity indicating the existence of an Fe-S cluster in the protein. In the current study we expressed human IRF in insect cells from recombinant baculovirus and analysed IRE-binding and aconitase activities under various culture conditions. Newly made apoprotein, synthesized in the absence of iron, was fully active in IRE-binding, but showed no aconitase activity. In contrast, IRF made by cells grown in high iron medium bound RNA poorly, but exhibited high aconitase activity with a Km of 9.2 microM for cis-aconitate. Apo-IRF was converted in vitro to active aconitase by Fe-S cluster-generating conditions, and under the same conditions lost its RNA-binding capacity. These results indicate that the two activities are mutually exclusive and controlled through formation of the Fe-S cluster.

Isolation of 2,4-Diacetylphloroglucinol from a Fluorescent Pseudomonad and Investigation of Physiological Parameters Influencing Its Production

Pseudomonas sp. strain F113 was isolated from the rhizosphere of sugar beets and shown to inhibit a range of plant pathogenic fungi by producing an antibioticlike compound. An antibiotic-negative mutant strain, F113G22, was generated by transposon mutagenesis. This mutant has lost the ability to inhibit both bacterial and fungal microorganisms on high-iron medium. The antibioticlike compound was subsequently identified as 2,4-diacetylphloroglucinol (DAPG), and a high-pressure liquid chromatographic assay was developed for to detect it quantitatively in growth culture media and soil. The growth temperature had a direct bearing on DAPG production by strain F113, with maximum production at 12 degrees C. The iron concentration, pH, and oxygen had no influence on DAPG production by strain F113 under the assay conditions used. However, a low ratio of culture volume to surface area available to the microbe in the growth container was critical for optimum DAPG production. Different types of carbon sources influenced DAPG production by strain F113 to various degrees. For example, sucrose, fructose, and mannitol promoted high yields of DAPG by strain F113, whereas glucose and sorbose resulted in very poor DAPG production.