Temperature inhibition of siderophore production in Azospirillum brasilense.

The effect of growth at 42 degrees C on the different components of the siderophore-mediated iron transport that are induced by iron limitation in Azospirillum brasilense was examined. Biosynthesis of the siderophore spirilobactin was strongly inhibited (20-fold) by growth at 42 degrees C, whereas the transport of iron by the ferric-spirilobactin transport system and the induction of the iron-regulated outer membrane proteins were unaffected.

Co-ordinated expression of the components of iron transport (mycobactin, exochelin and envelope proteins) in Mycobacterium neoaurum.

Mycobacterium neoaurum was grown with a range of iron concentrations from 0.01 to 4.0 micrograms/ml. Synthesis of the extracellular siderophore, exochelin, the intracellular iron storage compound, mycobactin and the iron-repressible envelope proteins were co-ordinately expressed. All three components of the iron transport system were synthesized when low amounts of iron (0.01 to 0.2 micrograms/ml) were added to the medium and were repressed when the iron concentration was increased to 0.5 micrograms/ml and above. These results re-inforce the conclusion that the iron-regulated proteins do fulfil an essential function in iron metabolism.

Analysis of ferrichrome biosynthesis in the phytopathogenic fungus Ustilago maydis: cloning of an ornithine-N5-oxygenase gene.

By using a non-enterobactin-producing enb-7 mutant of Salmonella typhimurium LT2 as a biological indicator, a novel screening method was developed for identifying mutants of Ustilago maydis defective in the biosynthesis of the siderophores ferrichrome and ferrichrome A. Two classes of siderophore mutations, both recessive, were isolated after mutagenesis of haploid cells of the corn smut fungus. Class I mutants no longer produced ferrichrome while retaining the ability to produce ferrichrome A; class II mutants were defective in the production of both ferrichrome and ferrichrome A. Genetic and biochemical data suggest that class II mutants are defective in the ability to hydroxylate L-ornithine to delta-N-hydroxyornithine, the first step in the biosynthesis of these siderophores. A genomic library of wild-type U. maydis DNA was constructed in the cosmid transformation vector pCU3, which contains a dominant selectable marker for hygromycin B resistance. Two cosmids, pSid1 and pSid2, were identified in this library by their ability to complement class II siderophore auxotrophs. The production of both siderophores was concomitantly restored in the majority of the resultant transformants. Transforming DNA could be recovered from the fungal, cosmid-containing transformants by in vitro packaging with lambda bacteriophage extracts. Alternatively, the clones could be identified by a sib selection procedure. Cotransformation was found to occur at a high frequency in the fungus and was used to determine that a 2.5-kilobase HindIII-NruI fragment in pSid1 was responsible for complementing the class II siderophore biosynthetic mutation.

Amonabactin, a novel tryptophan- or phenylalanine-containing phenolate siderophore in Aeromonas hydrophila.

Aeromonas hydrophila 495A2 excreted two forms of amonabactin, a new phenolate siderophore composed of 2,3-dihydroxybenzoic acid, lysine, glycine, and either tryptophan (amonabactin T) or phenylalanine (amonabactin P). Supplementing cultures with L-tryptophan (0.3 mM) caused exclusive synthesis of amonabactin T, whereas supplements of L-phenylalanine (0.3 to 30 mM) gave predominant production of amonabactin P. The two forms of amonabactin were separately purified by a combination of production and polyamide column chromatographic methods. Both forms were biologically active, stimulating growth in iron-deficient medium of an amonabactin-negative mutant. Of 43 additional siderophore-producing isolates of the Aeromonas species that were tested, 76% (19 of 25) of the A. hydrophila isolates were amonabactin positive, whereas only 19% (3 of 16) of the A. sobria isolates and all (3 of 3) of the A. caviae isolates produced amonabactin, suggesting a predominant synthesis of amonabactin in certain Aeromonas species.

[Direct screening of antibiotics-siderophores of bacterial origin]. / Napravlennyi skrining antibiotikov-sideroforov bakterial'nogo proiskhozhdeniia.

Antifungal activity of 275 strains belonging to 15 species of Pseudomonas was studied with using media containing no iron or supplemented with 100 micrograms/ml of FeCl3. 33 per cent of the cultures showed lower activity against phytopathogenic fungi in the presence of iron. Addition of this element did not influence the antifungal activity of phenazin and floroglucin derivatives isolated from Pseudomonas cultures. However, its addition markedly lowered the antifungal effect of some crude antibiotics and fluorescent pigments. A scheme for screening siderophore antibiotics with using Pseudomonas cultures is described.

Virulence properties and enterotoxin production of Aeromonas strains isolated from fish.

The biological activities in vivo and in vitro of 59 motile Aeromonas spp. isolated from fish and water tanks were simultaneously analyzed in poikilothermic and homoiothermic systems. A total of 64.3% of the isolates tested were pathogenic for fish, and 62% of Aeromonas hydrophila and A. sobria isolates either virulent or nonvirulent for fish were enterotoxigenic. Although the majority of the strains were proteolytic and amylolytic and produced DNase, other activities, such as elastase and staphylolysis, were only present in A. hydrophila. Most of the strains (96%) produced hemolysins, and 68% had agglutinating capacity, but neither isolates pathogenic for fish nor enterotoxigenic isolates showed specificity for trout or human erythrocytes, respectively. The production of siderophores, agglutination in acriflavine, and precipitation after boiling were found not to be useful tests for screening virulent strains. Although statistical analysis revealed a significant relationship between virulence for fish and positive results for arabinose and sucrose fermentations, elastase, and hemolysis of human erythrocytes, only lysine decarboxylase showed a significant positive relationship with enterotoxigenicity. Using extracellular products from representative Aeromonas strains with different virulence markers and belonging to distinct O serogroups, we demonstrated a lack of correlation between cytotoxicity for fish and homoiothermic cell lines and pathogenicity. The extracellular products from selected pathogenic A. hydrophila strains were lethal for rainbow trout and displayed proteolytic, hemolytic, and cytotoxic activities which were simultaneously lost after heat treatment. The findings reported here indicate that it is not possible to establish a common and single mechanism involved in the invasion of Aeromonas spp. in poikilothermic and homoiothermic hosts.

Siderophore production by Pasteurella multocida.

Pasteurella multocida grown under conditions of iron deprivation secreted into the culture medium a growth-enhancing factor which functioned as a siderophore. The siderophore was found to be neither a phenolate nor a hydroxamate by chemical tests and bioassays and was given the trivial name multocidin. Multocidin was partially purified and found to be a highly polar, nonaromatic, and dialyzable compound. This is the first report demonstrating the production of a siderophore by P. multocida.

Correlation of the virulence of Klebsiella pneumoniae K1 and K2 with the presence of a plasmid encoding aerobactin.

Nine isolates of Klebsiella pneumoniae belonging to capsular serotypes K1 and K2 were assayed for virulence in mice. Virulent isolates (50% lethal dose of less than 10(3) microorganisms) and avirulent isolates (50% lethal dose of over 10(6) microorganisms) were selected. Supplementation of a defined minimal medium with transferrin markedly reduced the growth of avirulent strains but had no significant effect on the growth of virulent strains. All isolates produced enterochelin, but only production of aerobactin could be correlated with virulence. The genes encoding aerobactin and its receptor protein were located on a 180-kilobase plasmid. They were cloned into the mobilizable vector pSUP202. Homology was demonstrated with the aerobactin operon of the Escherichia coli plasmid pColV-K30. Transfer of the recombinant plasmid pKP4 into an avirulent recipient enhanced virulence by 100-fold. These experiments demonstrated that aerobactin is an essential factor of pathogenicity in K. pneumoniae.

Influence of iron on growth, morphology, outer membrane protein composition, and synthesis of siderophores in Campylobacter jejuni.

Three human isolates of Campylobacter jejuni were grown in a biphasic culture medium with and without the addition of a synthetic chelator to induce iron limitation. Cells grown in low-iron medium exhibited slower growth rates and altered cellular morphology. Increased numbers of longer, more filamentous forms were seen in Gram-stained smears. Three proteins, with apparent Mrs of 82,000, 76,000, and 74,000, were consistently present in the outer membrane of cells grown in low-iron medium. At least one of these proteins (76,000 to 74,000) was exposed on the cell surface. A bioassay was used to look for the production of siderophores by these and other strains of C. jejuni. Seven of 26 strains tested produced detectable amounts of siderophores. Growing strains at 42 degrees C failed to suppress siderophore synthesis or to alter the outer membrane protein profiles of iron-starved cells. The ability of three strains to utilize exogenously supplied siderophores for growth in low-iron medium was also examined. All three strains were able to utilize enterochelin and ferrichrome, but none utilized aerobactin, rhodotorulic acid, or desferrioxamine B. The effect of iron on the virulence of C. jejuni for 11-day-old chicken embryos inoculated via the chorioallantoic membrane was also determined.

Hydroxamate production by Aquaspirillum magnetotacticum.

Spent culture fluids from Aquaspirillum magnetotacticum MS-1 grown at high (20 microM) but not low (5 microM) iron concentration contained material yielding a positive hydroxamate test. Cells possessed six major outer membrane proteins. Three outer membrane proteins ranging from 72,000 to 85,000 daltons were coordinately produced at iron concentrations conducive to hydroxamate production. A 55,000-dalton iron-repressible outer membrane protein was also present in strain MS-1 cultured at low but not high ferric quinate concentration. Culture fluids from strain MS-1 which were hydroxamate positive augmented growth of a Salmonella typhimurium siderophore-deficient (enb-7) mutant in low-iron medium, suggesting a role of hydroxamate in uptake of iron by the cell.

Iron uptake processes in Mycobacterium vaccae R877R, a mycobacterium lacking mycobactin.

The production of exochelins (MV) was established in Mycobacterium vaccae R877R under iron-deficient conditions in concentrations about five times greater than in Mycobacterium smegmatis. M. vaccae does not produce mycobactin nor is salicylic acid secreted into the medium. A simple method is described using 55Fe-labelled culture filtrates for assessing exochelin production and which would be applicable to other mycobacteria. One of the exochelins produced (MV3) is part of an active iron uptake system and another (MV1) is responsible for a passive uptake system. MV3 exochelin has similar chromatographic properties and biological activity to the major exochelin produced by M. smegmatis: iron uptake from MV3 exochelin was inhibited by dinitrophenol, NaN3 and HgCl2, and was judged to be an active transport process. This process was not inhibited by equimolar amounts of ferri-salicylate or ferri-citrate both of which could be used separately as sources of iron for the organism. Uptake from these latter sources was insensitive to metabolic inhibitors and uncouplers. The multiplicity of pathways for iron uptake in a single organism is discussed.

Relation between the hemolytic property and iron metabolism in Escherichia coli.

The hemolytic activity of wild-type strains of Escherichia coli was measured by a standardized method in liquid broth. The system also allowed us to investigate the influence of various Fe3+ concentrations in the cultures on the amount of secreted hemolysin. We found that the hemolysin secretion of all strains was clearly reduced after addition of FeCl3. However, the influence of additional iron chelators showed remarkable differences. The hemolytic activity of Hly plasmid-containing strains isolated in Berne significantly increased. Most of the strains with a chromosomal hemolysin determinant showed a similar effect, but to a lesser degree. Contrary to this, Hly plasmid-containing strains isolated in Essex and some strains with chromosomal hemolysin determinant were either not affected or even showed reduced hemolysin secretion after limitation of free iron ions in the broth. Our results suggest that hemolysin secretion in E. coli is related to the bacterial iron metabolism, and hemolysin secretion is differentially regulated among E. coli strains.

Isolation and analysis of genes involved in siderophore biosynthesis in plant-growth-stimulating Pseudomonas putida WCS358.

The plant-growth-stimulating Pseudomonas putida WCS358 was mutagenized with transposon Tn5. The resulting mutant colony bank was screened for mutants defective in the biosynthesis of the fluorescent siderophore. A total of 28 mutants, divided into six different classes, were isolated that were nonfluorescent or defective in iron acquisition or both. These different types of mutants together with the probable overall structure of the siderophore, i.e., a small peptide chain attached to a fluorescing group, suggest a biosynthetic pathway in which the synthesis of the fluorescing group is preceded by the synthesis of the peptide part. A gene colony bank of P. putida WCS358 was constructed with the broad-host-range cosmid vector pLAFR1. This genomic library, established in Escherichia coli, was mobilized into the 28 individual mutants, screening for transconjugants restored in fluorescence or growth under iron-limiting conditions or both. A total of 13 cosmids were found to complement 13 distinct mutants. The complementation analysis revealed that at least five gene clusters, with a minimum of seven genes, are needed for siderophore biosynthesis. Some of these genes seem to be arranged in an operon-like structure.

Effect of temperature on siderophore production by Candida albicans.

The purpose of this study was to examine the effect of elevated temperature on growth and siderophore production by Candida albicans. The results showed that an increase in incubation temperature from 37 degrees C to 41 degrees C produced a marked decrease in both the rate and quantity of siderophore production. Elevated temperature was unable to suppress growth of C. albicans in either a control culture medium or a deferrated culture medium. A significant suppression of growth compared to the controls was observed in the deferrated media at both 37 degrees C and 41 degrees C. However with time, the growth of cells in the deferrated media showed partial recovery which was followed by an increase in siderophore production. Thus, elevation of temperature to suppress growth and siderophore production by C. albicans appears to be an ineffective host defense mechanism.

Plasmids mediating iron uptake in Vibrio anguillarum strains isolated from turbot in Spain.

Vibrio strains isolated from diseased turbot in an experimental fish farm on the Atlantic coast of northwest Spain were identified as Vibrio anguillarum. The isolates shared many biochemical characteristics with V. anguillarum strains obtained from other sources, and harboured a plasmid species that showed extensive homology with plasmid pJM1, carried by V. anguillarum strain 775 isolated from an epizootic in North America. Restriction endonuclease analysis showed that the two plasmids were very similar albeit not identical. The presence of the plasmid in the turbot isolates was associated with their ability to cause disease in fish. Plasmid-carrying bacteria could also grow under conditions of iron limitation. Two outer membrane proteins, of 86 and 79 kDal, were induced, and a similar siderophore activity to that produced by V. anguillarum 775 was also detected under these conditions. The 86 kDal outer membrane protein cross-reacted immunologically with antiserum raised against the outer membrane protein OM2 produced by strain 775. Nonvirulent plasmidless derivatives were unable to grow under iron-limiting conditions, and were also unable to produce either siderophore activity or the 86 kDal outer membrane protein, suggesting the plasmid-mediated nature of these components.

Siderophore production by the pathogenic yeast, Candida albicans.

Biochemical assays were used to determine that some strains of Candida albicans were capable of simultaneous secretion of both the hydroxamate and phenolate-type siderophores when grown in a deferrated medium at 37 degrees C. All isolants of C. albicans released hydroxamate-type siderophores into the culture medium; whereas, approximately 40% of the strains simultaneously secreted phenolate-type siderophores. The presence of phenolate and hydroxamate-type siderophores in the culture medium was further confirmed by assaying the culture media with type specific siderophore-dependent bacterial auxotrophs. This is the first report showing production of both classes of siderophores by a pathogenic yeast.

Effect of subinhibitory concentrations of cephalosporins on surface properties and siderophore production in iron-depleted Klebsiella pneumoniae.

Subinhibitory MICs (sub-MICs) of several cephalosporins significantly reduced the enterochelin production of Klebsiella pneumoniae 327 grown under iron-depleted conditions and also reduced capsule formation regardless of iron availability. The surface hydrophobicity of K. pneumoniae 327 increased significantly when the bacteria were grown in either iron-sufficient or iron-depleted media in the presence of sub-MICs of all the cephalosporins used in this study. Antisera raised against a non-encapsulated K. pneumoniae strain caused rapid agglutination of K. pneumoniae 327 grown in the presence of sub-MICs of the cephalosporins but no agglutination of the same strain grown in drug-free media. The results indicated that the cephalosporins reduced enterochelin production and also capsule formation to the extent that noncapsular surface antigens were exposed, with possible significant consequences in vivo.

Highly toxinogenic but avirulent Park-Williams 8 strain of Corynebacterium diphtheriae does not produce siderophore.

The highly toxinogenic Park-Williams 8 strain of Corynebacterium diphtheriae grows slowly in vitro and is avirulent. C. diphtheriae Park-Williams 8 is defective in iron uptake and does not produce the corynebacterial siderophore corynebactin. Addition of partially purified corynebactin stimulated iron uptake and growth of iron-deprived C. diphtheriae Park-Williams 8 cells.