Characterization of a high-affinity iron transport system in Acinetobacter baumannii.

Analysis of a clinical isolate of Acinetobacter baumannii showed that this bacterium was able to grow under iron-limiting conditions, using chemically defined growth media containing different iron chelators such as human transferrin, ethylenediaminedi-(o-hydroxyphenyl)acetic acid, nitrilotriacetic acid, and 2,2'-bipyridyl. This iron uptake-proficient phenotype was due to the synthesis and secretion of a catechol-type siderophore compound. Utilization bioassays using the Salmonella typhimurium iron uptake mutants enb-1 and enb-7 proved that this siderophore is different from enterobactin. This catechol siderophore was partially purified from culture supernatants by adsorption chromatography using an XAD-7 resin. The purified component exhibited a chromatographic behavior and a UV-visible light absorption spectrum different from those of 2,3-dihydroxybenzoic acid and other bacterial catechol siderophores. Furthermore, the siderophore activity of this extracellular catechol was confirmed by its ability to stimulate energy-dependent uptake of 55Fe(III) as well as to promote the growth of A. baumannii bacterial cells under iron-deficient conditions imposed by 60 microM human transferrin. Polyacrylamide gel electrophoresis analysis showed the presence of iron-regulated proteins in both inner and outer membranes of this clinical isolate of A. baumannii. Some of these membrane proteins may be involved in the recognition and internalization of the iron-siderophore complexes.     

Iron-binding compounds and related outer membrane proteins in Vibrio cholerae non-O1 strains from aquatic environments

A total of 156 strains of Vibrio cholerae non-O1 from aquatic origins were examined for the presence of iron uptake mechanisms and compared with O1 strains and other Vibrio species. All non-O1 strains were able to grow in iron-limiting conditions, with MICs of ethylenediaminedi (O-hydroxyphenylacetic acid) ranging from 20 microM to 2 mM. The production of siderophores was demonstrated by growth in chrome azurol S agar and cross-feeding assays. All strains produced phenolate-type compounds, as assessed by the chemical tests and by bioassays with Salmonella typhimurium enb-7. Some of the strains also promoted the growth of S. typhimurium enb-1 (which can use only enterobactin as a siderophore) as well as some strains of Vibrio anguillarum deficient in the anguibactin-mediated system. The chromatographic analyses and absorption spectra of siderophores extracted from culture supernatants suggest that vibriobactin may be produced by the strains examined. Interestingly, some strains also produced hydroxamate-type compounds, as determined by chemical tests, and were able to promote the growth of an aerobactin-deficient strain of Escherichia coli. These results were confirmed by the absorption spectra and chromatographic analyses of the culture extracts. The synthesis of iron-regulated outer membrane proteins in representative strains was also examined. The molecular sizes of the main induced proteins ranged from 70 to 78 kilodaltons. These results indicate that several iron uptake mechanisms which could be involved in environmental survival and pathogenicity are present in environmental V. cholerae non-O1 strains.     

Multiple alpha-keto aciduria in Microtus montanus chronically infected with Trypanosoma brucei gambiense

Microtus montanus chronically infected with a monomorphic strain of Trypanosoma brucei gambiense excreted in urine greatly elevated quantities of not only the aromatic alpha-keto acids, phenylpyruvic and 4-hydroxyphenylpyruvic acids, but also two aliphatic alpha-keto acids, pyruvic and alpha-ketoglutaric acids. Elevated keto acid excretion began approximately midway through infection and quantities remained elevated until death. Daily keto acid excretion did not correlate with daily parasitemia. Thus, a large metabolic disturbance exists in laboratory animals infected with African trypanosomes. The multiple alpha-keto aciduria potentially contributes to the pathogenesis of chronic African trypanosomiasis.     

Iron-binding compounds and related outer membrane proteins in Vibrio cholerae non-O1 strains from aquatic environments.

A total of 156 strains of Vibrio cholerae non-O1 from aquatic origins were examined for the presence of iron uptake mechanisms and compared with O1 strains and other Vibrio species. All non-O1 strains were able to grow in iron-limiting conditions, with MICs of ethylenediaminedi (O-hydroxyphenylacetic acid) ranging from 20 microM to 2 mM. The production of siderophores was demonstrated by growth in chrome azurol S agar and cross-feeding assays. All strains produced phenolate-type compounds, as assessed by the chemical tests and by bioassays with Salmonella typhimurium enb-7. Some of the strains also promoted the growth of S. typhimurium enb-1 (which can use only enterobactin as a siderophore) as well as some strains of Vibrio anguillarum deficient in the anguibactin-mediated system. The chromatographic analyses and absorption spectra of siderophores extracted from culture supernatants suggest that vibriobactin may be produced by the strains examined. Interestingly, some strains also produced hydroxamate-type compounds, as determined by chemical tests, and were able to promote the growth of an aerobactin-deficient strain of Escherichia coli. These results were confirmed by the absorption spectra and chromatographic analyses of the culture extracts. The synthesis of iron-regulated outer membrane proteins in representative strains was also examined. The molecular sizes of the main induced proteins ranged from 70 to 78 kilodaltons. These results indicate that several iron uptake mechanisms which could be involved in environmental survival and pathogenicity are present in environmental V. cholerae non-O1 strains.     

High-performance liquid chromatographic analysis of alpha-keto acids produced from amino acid metabolism in oral Bacteroides

Profiles of metabolic alpha-keto acids were determined by a high-performance liquid chromatographic method and applied to characterization of oral black-pigmented Bacteroides. Each bacterial strain was incubated with amino acids in a chemically defined medium. After production alpha-keto acids were purified by hydrazide gel column treatment and converted to u.v.-absorbing derivatives. They were analysed by reversed-phase ion-pair chromatography. Black-pigmented Bacteroides species were differentiated into two groups according to production of aromatic alpha-keto acids. Bacteroides gingivalis, B. endodontalis and B. loescheii produced both p-hydroxyphenylpyruvic and phenylpyruvic acids. However, no such alpha-keto acids were produced by B. levii, B. intermedius and B. denticola. In addition, production profiles of several aliphatic alpha-keto acids (alpha-ketoglutaric, pyruvic, alpha-ketobutyric, alpha-ketoisovaleric, alpha-ketoisocaproic, and alpha-keto-beta-methylvaleric acids) separated each individual species in such groups. The present study offers useful chemotaxonomic information on amino acid metabolic activity of oral black-pigmented Bacteroides species.     

Alpha-keto acids are novel siderophores in the genera Proteus, Providencia, and Morganella and are produced by amino acid deaminases.

Growth promotion and iron transport studies revealed that certain alpha-keto acids generated by amino acid deaminases, by enterobacteria of the Proteus-Providencia-Morganella group (of the tribe Proteeae), show significant siderophore activity. Their iron-binding properties were confirmed by the chrome azurol S assay and UV spectra. These compounds form ligand-to-metal charge transfer bands in the range of 400 to 500 nm. Additional absorption bands of the enolized ligands at 500 to 700 nm are responsible for color formation. Siderophore activity was most pronounced with alpha-keto acids possessing an aromatic or heteroaromatic side chain, like phenylpyruvic acid and indolylpyruvic acid, resulting from deamination of phenylalanine and tryptophan, respectively. In addition, alpha-keto acids possessing longer nonpolar side chains, like alpha-ketoisocaproic acid or alpha-ketoisovaleric acid and even alpha-ketoadipic acid, also showed siderophore activity which was absent or negligible with smaller alpha-keto acids or those possessing polar functional groups, like pyruvic acid, alpha-ketobutyric acid, or alpha-ketoglutaric acid. The fact that deaminase-negative enterobacteria, like Escherichia coli and Salmonella spp., could not utilize alpha-keto acids supports the view that specific iron-carboxylate transport systems have evolved in members of the tribe Proteeae and are designed to recognize ferric complexes of both alpha-hydroxy acids and alpha-keto acids, of which the latter can easily be generated by L-amino acid deaminases in an amino acid-rich medium. Exogenous siderophores, like ferric hydroxamates (ferrichromes) and ferric polycarboxylates (rhizoferrin and citrate), were also utilized by members of the tribe Proteeae.     

Stereoselectivity and stereospecificity of the alpha,beta-dihydroxyacid dehydratase from Salmonella typhimurium.

1. In addition to the known 2R,3R- and 2R, 3S-2,3-dihydroxy-3-methylpentanoic acids (DHI), the 1S,3S- and sS,DR-isomers were prepared. 2S-2,3-Dihydroxy-3-methylbutanoic acid (DHV) was also prepared in addition to the known 2R-isomer. 2. The six dihydroxy acids were examined for their ability to promote the growth of isoleucine-valine (ilv)-requiring strains of Salmonella typhimurium and to serve as substrates for the alpha,beta-dihydroxyacid dehydratase of the same organism. 3. Only 2R,3R-2,3-dihydroxy-3-methylpentanoic and 2R-2,3-dihydroxy-3-methylbutanoic acids supported growth of the ilv strains of S. typhimurium. 4. alpha,beta-Dihydroxyacid dehydratase utilized the three isomers with the 2R-configuration as substrates but not those with the 2S-configuration. 5. In an additional growth study that utilized the 3R- and 3S-isomers of 3-methyl-2-oxopentanoic acid, the alpha-keto acid analogue of isoleucine, only the 3S-isomer supported growth. 6. It is concluded that the mechanism of action of the dehydratase is stereospecific in that the proton that is attached to C-3 of the substrate occupies the same steriochemical position as the departing hydroxyl group (Fig. 6).     

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.     

Induction of porphyrin synthesis in etiolated bean leaves by chelators of iron

Primary leaves of 7- to 9-day-old etiolated seedlings of Phaseolus vulgaris L. var. Red Kidney infiltrated in darkness with aqueous solutions of alpha, alpha'-dipyridyl, o-phenanthroline, pyridine-2-aldoxime, pyridine-2-aldehyde, 8-hydroxyquinoline, or picolinic acid synthesize large amounts of magnesium protoporphyrin monomethyl ester and lesser amounts of magnesium protoporphyrin, protoporphyrin, and protochlorophyllide. Pigment formation proceeds in a linear manner for up to 21 hours after vacuum infiltration with 10 mm alpha, alpha'-dipyridyl. Etiolated tissues of Zea mays L., Cucumis sativus L., and Pisum sativum L. respond in the same way to dipyridyl treatment. Compounds active in eliciting this response are aromatic heterocyclic nitrogenous bases which also act as bidentate chelators and form extremely stable complexes with iron; other metal ion chelators, such as ethylenediaminetetraacetic acid, salicylaldoxime, and sodium diethyldithiocarbamate, do not elicit any pigment synthesis. The ferrous, ferric, cobaltous, and zinc chelates of alpha, alpha'-dipyridyl are similarly ineffective. If levulinic acid is supplied to etiolated bean leaves together with alpha, alpha'-dipyridyl, porphyrin production is inhibited and delta-aminolevulinic acid accumulates in the tissue. Synthesis of porphyrins proceeds in the presence of 450 micrograms per milliliter chloramphenicol or 50 micrograms per milliliter cycloheximide with only partial diminution. We propose that heme or an iron-protein complex blocks the action of the enzyme(s) governing the synthesis of delta-aminolevulinic acid in etiolated leaves in the dark and that iron chelators antagonize this inhibition, leading to the biosynthesis of delta-aminolevulinic acid and porphyrins.     

Inhibition of glycine oxidation by pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in rat liver mitochondria: presence of interaction between the glycine cleavage system and alpha-keto acid dehydrogenase complexes

Pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids which were transaminated products of valine, leucine, and isoleucine inhibited glycine decarboxylation by rat liver mitochondria. However, glycine synthesis (the reverse reaction of glycine decarboxylation) was stimulated by those alpha-keto acids with the concomitant decarboxylation of alpha-keto acid added in the absence of NADH. Both the decarboxylation and the synthesis of glycine by mitochondrial extract were affected similarly by alpha-ketoglutarate and branched-chain alpha-keto acids in the absence of pyridine nucleotide, but not by pyruvate. This failure of pyruvate to have an effect was due to the lack of pyruvate oxidation activity in the mitochondrial extract employed. It indicated that those alpha-keto acids exerted their effects by providing reducing equivalents to the glycine cleavage system, possibly through lipoamide dehydrogenase, a component shared by the glycine cleavage system and alpha-keto acid dehydrogenase complexes. On the decarboxylation of pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in intact mitochondria, those alpha-keto acids inhibited one another. In similar experiments with mitochondrial extract, decarboxylations of alpha-ketoglutarate and branched-chain alpha-keto acid were inhibited by branched-chain alpha-keto acid and alpha-ketoglutarate, respectively, but not by pyruvate. NADH was unlikely to account for the inhibition. We suggest that the lipoamide dehydrogenase component is an indistinguishable constituent among alpha-keto acid dehydrogenase complexes and the glycine cleavage system in mitochondria in nature, and that lipoamide dehydrogenase-mediated transfer of reducing equivalents might regulate alpha-keto acid oxidation as well as glycine oxidation.     

Regulation of synthesis of GTP-cyclohydrolase participating in yeast falvinogenesis by iron

Pichia guilliermondii, Schwanniomyces occidentalis, Torulopsis candida and several riboflavin-dependent mutants of Torulopsis candida were grown in a medium with a low concentration of iron. In these conditions, the activity of GTP-cyclohydrolase which catalyzes the first step of flavinogenesis increases. The activity of the enzyme increases also when the cells of T. candida and P. guilliermondii with a high content of iron are incubated with alpha, alpha'-dipyridyl which induces overproduction of riboflavin; this action of alpha, alpha'-dipyridyl is eliminated by cycloheximide. Therefore, iron deficiency in the cells of these yeasts causes derepression of GTP-cyclohydrolase participating in riboflavin biosynthesis. The activity of the enzyme is inhibited by FAD but not by FMN and riboflavin.     

Proteus mirabilis amino acid deaminase: cloning, nucleotide sequence, and characterization of aad.

Proteus, Providencia, and Morganella species produce deaminases that generate alpha-keto acids from amino acids. The alpha-keto acid products are detected by the formation of colored iron complexes, raising the possibility that the enzyme functions to secure iron for these species, which do not produce traditional siderophores. A gene encoding an amino acid deaminase of uropathogenic Proteus mirabilis was identified by screening a genomic library hosted in Escherichia coli DH5 alpha for amino acid deaminase activity. The deaminase gene, localized on a cosmid clone by subcloning and Tn5::751 mutagenesis, was subjected to nucleotide sequencing. A single open reading frame, designated aad (amino acid deaminase), which appears to be both necessary and sufficient for deaminase activity, predicts a 473-amino-acid polypeptide (51,151 Da) encoded within an area mapped by transposon mutagenesis. The predicted amino acid sequence of Aad did not share significant amino acid sequence similarity with any other polypeptide in the PIR or SwissProt database. Amino acid deaminase activity in both P. mirabilis and E. coli transformed with aad-encoding plasmids was not affected by medium iron concentration or expression of genes in multicopy in fur, cya, or crp E. coli backgrounds. Enzyme expression was negatively affected by growth with glucose or glycerol as the sole carbon source but was not consistent with catabolite repression.     

Siderophore production by Aeromonas salmonicida.

Growth under conditions of iron-restriction and the production of siderophores was examined in 21 typical and 14 atypical strains of Aeromonas salmonicida. With the exception of one atypical strain, all strains grew and multiplied in the presence of the high-affinity iron chelators ethylenediamine di(o-hydroxyphenylacetic acid), alpha, alpha'-dipyridyl or transferrin. Chrome azurol S agar was used to screen bacterial strains growing under these conditions for the production of siderophores. Siderophore production was detected only in the typical strains. Siderophores were also detected in the iron-restricted culture supernatants of typical strains. Siderophores were also detected in the iron-restricted culture supernatants of typical strains, where they were associated with an iron-binding activity. The siderophore was extracted from iron-restricted culture supernatant of one strain by adsorption onto an XAD-7 resin; it behaved as a 2,3-diphenol-catechol in several colorimetric assays. The results indicate that although both typical and atypical strains of A. salmonicida grow and multiply under conditions of iron-restriction, they use different iron-uptake mechanisms, siderophore-mediated and siderophore-independent, respectively. In cross-feeding assays, growth of typical strains was stimulated only by homologous iron-restricted supernatant, suggesting strain differences in the siderophore produced. However, one strain produced a culture supernatant with growth-stimulating activity for other typical and also atypical strains.     

Production of organic acids by bacteria isolated from soil, rhizosphere and mycorrhizosphere of pine (Pinus sylvestris L.)

The bacteria studied released into the medium ten to eleven organic acids. Soil organisms excreted mainly pyruvic and alpha-ketoglutaric acids, while strains from the root zone--gluconic acid and unidentified uronic acid (y2). Mean indices of total production of the organic acids by bacteria were in the following order: rhizosphere less than soil less than mycorrizosphere. Bacteria from the root zone released into the medium very large amounts of pyruvic, gluconic, and uronic (y2) acids--in some instances several times higher than bacterial dry mass.     

Lactobacillus growth and membrane composition in the presence of linoleic or conjugated linoleic acid

Five Lactobacillus strains of intestinal and food origins were grown in MRS broth or milk containing various concentrations of linoleic acid or conjugated linoleic acid (CLA). The fatty acids had bacteriostatic, bacteriocidal, or no effect depending on bacterial strain, fatty acid concentration, fatty acid type, and growth medium. Both fatty acids displayed dose-dependent inhibition. All strains were inhibited to a greater extent by the fatty acids in broth than in milk. The CLA isomer mixture was less inhibitory than linoleic acid. Lactobacillus reuteri ATCC 55739, a strain capable of isomerizing linoleic acid to CLA, was the most inhibited strain by the presence of linoleic acid in broth or milk. In contrast, a member of the same species, L. reuteri ATCC 23272, was the least inhibited strain by linoleic acid and CLA. All strains increased membrane linoleic acid or CLA levels when grown with exogenous fatty acid. Lactobacillus reuteri ATCC 55739 had substantial CLA in the membrane when the growth medium was supplemented with linoleic acid. No association between level of fatty acid incorporation into the membrane and inhibition by that fatty acid was observed.     

Pathways of 4-hydroxybenzoate degradation among species of Bacillus.

The pathways used by three bacterial strains of the genus Bacillus to degrade 4-hydroxybenzoate are delineated. When B. brevis strain PHB-2 is grown on 4-hydroxybenzoate, enzymes of the protocatechuate branch of the beta-ketoadipate pathway are induced. In contrast, B. circulans strain 3 contains high levels of the enzymes of the protocatechuate 2,3-dioxygenase pathway after growth on 4-hydroxybenzoate. B. laterosporus strain PHB-7a degrades 4-hydroxybenzoate by a novel reaction sequence. After growth on 4-hydroxybenzoate, strain PHB-7a contains high levels of gentisate oxygenase (EC 1.13.11.4) and maleylpyruvate hydrolase. Whole cells of strain PHB-7a (grown on 4-hydroxylbenzoate) accumulate 2,5-dihydroxybenzoate (gentisate) from 4-hydroxybenzoate when incubated in the presence of 1mM alpha,alpha'-dipyridyl. Thus, strain PHB-7a appears to convert 4-hydroxybenzoate to gentisate, which is further degraded by the glutathione-independent gentisic acid pathway. These pathway delineations provide evidence that Bacillus species are derived from a diverse evolutionary background.     

Distribution of plasmid- and chromosome-mediated iron uptake systems in Vibrio anguillarum strains of different origins.

We investigated the incidence of plasmid-mediated and chromosome-mediated iron uptake systems in strains of Vibrio anguillarum that belong to serotypes O1 and O2 and were isolated from different fish species and in different geographic areas. All of the strains gave positive reactions in CAS agar medium and in the Arnow test, which indicated that catechol types of siderophores were produced. The majority of V. anguillarum serotype O1 strains harbored a 65-kb plasmid similar to plasmid pJM1 from strain 775, which encodes the siderophore anguibactin and its outer membrane receptor, protein OM2. All of the isolates harboring this plasmid promoted the growth of an anguibactin-deficient receptor-proficient mutant derived from strain 775, but none of these isolates promoted the growth of mutants lacking receptor OM2. Furthermore, under iron-limiting conditions all of these strains induced outer membrane proteins that were identical in size to protein OM2 of strain 775. In contrast, none of the serotype O2 strains contained a high-molecular-weight plasmid, but all of them induced the growth of mutants defective in the anguibactin-mediated system regardless of the presence or absence of receptor OM2. The serotype O2 strains, but not the plasmid-bearing serotype O1 strains, also induced the growth of Salmonella typhimurium enb-1 which utilizes only enterobactin as a siderophore.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of plasmid- and chromosome-mediated iron uptake systems in Vibrio anguillarum strains of different origins

We investigated the incidence of plasmid-mediated and chromosome-mediated iron uptake systems in strains of Vibrio anguillarum that belong to serotypes O1 and O2 and were isolated from different fish species and in different geographic areas. All of the strains gave positive reactions in CAS agar medium and in the Arnow test, which indicated that catechol types of siderophores were produced. The majority of V. anguillarum serotype O1 strains harbored a 65-kb plasmid similar to plasmid pJM1 from strain 775, which encodes the siderophore anguibactin and its outer membrane receptor, protein OM2. All of the isolates harboring this plasmid promoted the growth of an anguibactin-deficient receptor-proficient mutant derived from strain 775, but none of these isolates promoted the growth of mutants lacking receptor OM2. Furthermore, under iron-limiting conditions all of these strains induced outer membrane proteins that were identical in size to protein OM2 of strain 775. In contrast, none of the serotype O2 strains contained a high-molecular-weight plasmid, but all of them induced the growth of mutants defective in the anguibactin-mediated system regardless of the presence or absence of receptor OM2. The serotype O2 strains, but not the plasmid-bearing serotype O1 strains, also induced the growth of Salmonella typhimurium enb-1 which utilizes only enterobactin as a siderophore.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lactic Acid Utilization by the Cutaneous Micrococcaceae

Human cutaneous staphylococci and micrococci utilized lactic acid as an energy source on a minimal medium. Propionic acid was not utilized, but l(+)-lactic acid and pyruvic acid could replace ld-lactic acid as a substrate. Selected strains of cocci were inhibited more by the l(+) and d(-) forms of lactic acid than the balanced ld form, particularly at pH 5.6. With proper dilution of substrate, lactic acid was utilized by selected strains in the presence of 10 mug of oleic and palmitic acids per ml.     

Itoic Acid Synthesis in Bacillus subtilis

Under conditions of iron deficiency, strains of Bacillus subtilis produced 2,3-dihydroxybenzoic acid (DHB), 2,3-dihydroxybenzolyglycine (DHBG), or both of these compounds. DHB(G) production [production of DHB(G) refers to the production of DHB, or DHBG, or both] was proportional to the amount of iron present and occurred logarithmically, paralleling growth. Supplementation of media with more than 150 mug of iron per liter at zero-time inhibited DHB accumulation completely. In the presence of DHB, lower levels of iron inhibited DHB(G) production, so that the actual inhibitor of synthesis may involve the Fe(3+):[DHB(G)](3) complex. The strains producing DHBG also produced coproporphyrin III during iron-deficient growth, whereas a strain producing DHB did not produce coproporphyrin III under these conditions. Accumulation of DHB(G) was influenced by the levels of aromatic amino acids and anthranilic acid in the medium. In vivo experiments with strain B-1471 demonstrated that DHB was coupled to added glycine to form DHBG. Metabolism of DHB(G) was observed in two of the strains studied.