Characterisation of a siderophore from Acinetobacter calcoaceticus

Abstract The Gram-negatice bacterium Acinetobacter calcoaceticus was examined for production of siderophores and iron-repressible outer membrane proteins following growth in iron-restricted media. The iron chelator, 2,3-dihydroxybenzoic acid was identified in the culture supernatant bu ¹H nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). A group of outer membrane proteins between 80 and 85 kDa were induced under iron restriction.     

Influence of iron on growth and extracellular products of Acinetobacter baumannii

Iron is an important nutrient required by bacteria for optimal growth. Acquisition of iron from the host where iron is restricted is an important mediator of bacterial pathogenesis. In iron deplete chemically defined medium (CDM-Fe) growth of Acinetobacter baumannii was restricted as compared to iron replete medium (CDM + Fe). Bacteria developed four high molecular weight outer membrane proteins (OMPs) of 88, 84, 80 and 77 kDa in CDM-Fe medium which were absent in CDM + Fe medium, and are known iron regulated outer membrane proteins (IROMPs). A. baumannii secreted siderophores extracellularly into the medium which act as iron chelators which had been demonstrated in the supernatants of CDM-Fe media. The siderophore was of catechol type. This shows that A. baumannii under iron restricted conditions express IROMPs along with production of catechol type siderophore in order to acquire iron from the external milieu.     

Iron-Dependent Production of Hydroxamate by Sodium-Dependent Azotobacter chroococcum

The sodium-dependent strain 184 of Azotobacter chroococcum was unable to grow significantly in iron-limited medium, but did produce iron-repressible outer membrane proteins. Siderophores were not produced under these conditions. Citric acid was excreted, but not in response to iron limitation. This strain, however, was able to grow in insoluble mineral iron sources, and under these conditions the cells produced a hydroxamate. Growth on minerals and hydroxamate production was dependent on a low level of freely exchangeable iron. Optimal hydroxamate production was observed with 0.75 μM ferric citrate, and hydroxamate production was repressed by >5 μM iron. Despite this iron requirement, hyroxamate was only formed during internal iron limitation of the cells. Iron-containing cells were able to grow in iron-limited medium but only produced hydroxamate when their iron-per-cellular-protein content was low. These results, the spectral changes observed upon Fe³⁺ addition, and iron-uptake coincident with hydroxamate production suggested that the hydroxamate was a siderophore.     

Derepression of the Azotobacter vinelandii siderophore system, using iron-containing minerals to limit iron repletion

Azotobacter vinelandii solubilized iron from certain minerals using only dihydroxybenzoic acid, which appeared to be produced constitutively. Solubilization of iron from other minerals required dihydroxybenzoic acid and the siderophore N,N'-bis-(2,3- dihydroxybenzoyl )-L-lysine ( azotochelin ) or these chelators plus the yellow-green fluorescent siderophore azotobactin . In addition to this sequential production of siderophores, cells also demonstrated partial to hyperproduction relative to the iron-limited control. The iron sources which caused partial derepression of the siderophores caused derepression of all the high-molecular-weight iron-repressible outer membrane proteins except a 77,000-molecular-weight protein, which appeared to be coordinated with azotobactin production. Increased siderophore production correlated with increased production of outer membrane proteins with molecular weights of 93,000, 85,000, and 77,000, but an 81,000-molecular-weight iron-repressible protein appeared at a constant level despite the degree of derepression. When iron was readily available, it appeared to complex with a 60,000-molecular-weight protein believed to form a surface layer on the A. vinelandii cell.     

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.     

Zn Increases Siderophore Production in Azotobacter vinelandii

When Azotobacter vinelandii was grown in the presence of low levels of iron, the addition of 20 or 40 muM ZnSO(4) caused earlier production of the catechol siderophores and a dramatic increase in the amount of azotobactin. The level of cellular iron was not significantly lowered in Zn -grown cells, which suggested that Zn was not causing more severe, or earlier, iron limitation. Also, Zn did not appear to affect production of the high-molecular-weight outer membrane iron-repressible proteins that presumably function as ferrisiderophore receptors. Spectrophotometric examination of ion binding to the siderophores revealed that while the siderophores appeared to bind Zn, only in the case of azotochelin was iron unable to completely overcome any Zn -induced changes in the absorption spectra. This appeared to rule out direct competition of Zn with iron for binding to the siderophores. Fe uptake was depressed both in Zn -grown cells and in Zn -free cells to which Zn was added during the uptake assay, except with azotobactin, with which the level of Fe uptake by Zn -grown cells was close to control levels. These results suggested two possible sites where Zn could be acting, one involving the biosynthesis of siderophores and possibly the genetic regulation of the iron assimilation system and the other involving an internal point common to iron assimilation by both high- and low-affinity iron uptake.     

Iron-dependent production of a heat-modifiable, 23,000-Mr outer membrane protein in Paracoccus denitrificans.

Production of a 23,000-Mr major outer membrane protein of Paracoccus denitrificans ATCC 13543 was dependent upon the addition of iron to a succinate-salts medium. The 23,000-Mr protein was not produced in an iron-deficient medium, but production of five outer membrane proteins in the 85,000- to 72,000-Mr range and of catechol were induced. The 23,000-Mr protein was not produced in a complex medium even when ferric citrate was added to the medium. Production of the protein was influenced by the carbon source and was decreased by peptone.     

The DNA gyrase inhibitors,nalidixic acid and oxolinic acid,prevent iron-mediated repression of catechol siderophore synthesis inAzotobacter vinelandii

Summary Low concentrations of nalidixic acid and oxolinic acid that were just inhibitory toAzotobacter vinelandii growth promoted the production of the catechol siderophores azotochelin and aminochelin, in the presence of normally repressive concentrations of Fe³⁺. There was a limited effect on the pyoverdin siderophore, azotobactin, where low concentrations of Fe³⁺ were rendered less repressive, but the repression by higher concentrations of Fe³⁺ was normal. These drugs did not induce high-molecular-mass iron-repressible outer-membrane proteins and similar effects on the regulation of catechol siderophore synthesis were not produced by novobiocin, coumermycin, or ethidium bromide. The timing of nalidixic acid and Fe³⁺ addition to iron-limited cells was critical. Nalidixic acid had to be added before iron-repression of catechol siderophore synthesis and before the onset of iron-sufficient growth. Continued production of the catechol siderophores, however, was not due to interference with normal iron uptake. These data indicated that nalidixic acid prevented normal iron-repression of catechol siderophore synthesis but could not reverse iron repression once it had ocurred. The possible roles of DNA gyrase activity in the regulation of catechol siderophore synthesis is discussed.     

Iron-regulated outer membrane proteins and non-siderophore-mediated iron acquisition by Paracoccus denitrificans

Abstract Deprivation of Paracoccus denitrificans of iron in sodium molybdate-containing medium caused a slower rate of growth and lower final cell yield, in contrast to our previous studies in non-sodium molybdate-containing medium, where iron deprivation had little effect on growth rate. Five high M _r outer membrane proteins and catechol production were induced in iron-deprived cultures. The fifth protein, M _r 72 000, was produced later than the others. Growth of iron-deprived cells in medium containing 20 μM ferric citrate repressed siderophore and iron deprivation-induced protein production, and led to production of an M _r 23 000 outer membrane protein (half maximum production after 5 h). Synthesis of the M _r 23 000 and high M _r proteins appeared to be mutally exclusive, and to be regulated by the cell's iron status. Cells inoculated into medium containing 20 μM ferric citrate took up 92% of the iron within 1 h, suggesting the occurrence of a nonsiderophore mediated, 'low affinity' iron uptake pathway.     

Role of the lipB gene product in the folding of the secreted lipase of Pseudomonas glumae

The LipB protein of Pseudomonas glumae is essential for the production of active extracellular lipase encoded by the lipA gene. When lipase is overproduced in P. glumae in the absence of a functional lipB gene, the enzyme accumulates intracellularly in an inactive conformation. Heterologous expression of the lipase in Pseudomonas aeruginosa, Bacillus subtilis and Escherichia coli indicated that LipB is not directly involved in the trans location of the lipase across the inner or outer membrane. However, the presence of LipB was essential for obtaining active lipase and had a profound influence on the stability of the protein to proteolytic degradation. Inactive iipase, produced in the absence of LipB could be activated in vitro by unfolding and refolding, which demonstrates that LipB activity is not responsible for an essential covalent modification of the enzyme. We propose that LipB is a lipase-specific foldase. Furthermore, proper folding of the lipase in the periplasm appears to be essential for Xcp-mediated translocation across the outer membrane.     

Involvement of outer membrane proteins in enterochelin-mediated iron uptake in Escherichia coli.

Escherichia coli K-12 grown in iron-deficient media contained a large amount of outer membrane proteins O-2a, O-2b, and O-3, while cells grown in iron-supplemented media contained far smaller amounts of these proteins. The iron uptake by the iron-deficient cells was significantly stimulated in the presence of enterochelin, while that by the iron-rich cells was not. The outer membrane isolated from cells grown in the iron-deficient media showed enterochelin-stimulated binding of iron, while the outer membrane from iron-rich cells and cytoplasmic membranes from both types of cells did not show such binding activity. The amount of iron bound by the outer membrane was almost equivalent to the amount of O-2a, O2b, or O-3, irrespective of the amount of these proteins in the outer membrane, which is controlled by the amount of iron in the medium. Small particles rich in these proteins were prepared from cells by EDTA extraction. The particles were active in enterochelin-mediated iron binding and the amount of iron bound was equivalent to the amount of each of these proteins in the particles. Although the outer membrane of E. coli B was as active in iron binding as that of E. coli K-12, it did not possess an appreciable amount of O-2a. Gel electrophoretic analysis revealed that 9-2b and 9-3 were identical with the proteins missing mutants feuB and feuA, respectively.     

Characterization of Vibrio parahaemolyticus manganese-resistant mutants in reference to the function of the ferric uptake regulatory protein

In many bacteria, the ferric uptake regulatory protein (Fur) has a central role in the negative regulation of genes affected by iron limitation. In this study, Vibrio parahaemolyticus strains carrying mutations in the fur gene encoding Fur were isolated by the manganese selection method to assess the function of Fur in connection with alternations in the coordinate expression of the siderophore vibrioferrin (VF) and iron-repressible outer membrane proteins (IROMPs). Ten out of 25 manganese-resistant mutants constitutively produced VF and expressed at least two IROMPs irrespective of the iron concentration in the medium. PCR-direct DNA sequencing of the fur genes in these mutants identified four different point mutations causing amino acid changes. Moreover, a fur overexpressing plasmid was constructed to prepare antiserum against V. parahaemolyticus Fur. Western blotting with this antiserum revealed that the intracellular abundance of the wild-type Fur was not significantly affected by the iron concentrations in the growth medium, and that the Fur proteins of the mutant strains occurred at substantially smaller amounts and/or migrated more rapidly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the wild-type Fur. These data afford an additional insight into the structure-function relationship of Fur and imply its involvement in the iron acquisition systems of V. parahaemolyticus, although it is yet unknown whether its action on the target genes is direct or indirect.     

Characterization of a pyoverdine-deficient mutant of Pseudomonas fluorescens impaired in the secretion of extracellular lipase

A mutant of Pseudomonas fluorescens strain B52 deficient in the synthesis of the fluorescent pigment, pyoverdine, was isolated. Absence of pyoverdine and other siderophores was confirmed by gel filtration, a specific siderophore assay, and inhibition studies with the iron chelator EDDA. Both parent and mutant synthesized additional outer membrane proteins in response to iron-limitation. Mutant cells cultured in the absence of iron(III) accumulated ⁵⁵Fe-labeled pyoverdine. The mutant produced extracellular proteinase normally on various media, but was deficient in lipase secretion. Growth of the mutant with partially-purified pyoverdine resulted in a 2.5-fold stimulation of lipase secretion. The mutant grew poorly in deferrated medium; however, the addition of iron(III) stimulated growth. Proteinase secretion in deferrated medium was stimulated over a narrow range of iron(III) concentration, while lipase secretion was only slightly affected. The data suggest that separate regulatory mechanisms exist for the control of proteinase and lipase secretion by iron(III).Contribution No. 768 from the Food Research Centre     

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.     

INDUCTION OF SPECIFIC PROTEINS IN EUKARYOTIC ALGAE GROWN UNDER IRON-, PHOSPHORUS-, OR NITROGEN-DEFICIENT CONDITIONS1

What limits phytoplankton growth in nature? The answer is elusive because of methodological problems associated with bottle incubations and nutrient addition experiments. We are investigating the possibility that antibodies to proteins repressed by a specific nutrient can be used as probes to indicate which nutrient limits photosynthetic carbon fixation in the ocean. The diatom Phaeodactylum tricornutum Bohlin and the chlorophyte Dunaliella tertiolecta Butcher were grown in batch cultures in artificial seawater and f/2 nutrient lacking either phosphorus, iron, or nitrogen. Chlorosis was induced by nutrient limitation in both species with the exception of phosphorus-limited D. tertiolecta. The synthesis and appearance of specific proteins were followed by labeling with ¹⁴C-bicarbonate. Nutrient limitation in general leads to a decrease in the quantum efficiency of photosystem II, suggesting that deficiency of any nutrient affects the photosynthetic apparatus to some degree: however, the effect of nitrogen and iron limitation on quantum efficiency is more severe than that of phosphorus. A crude fractionation of the soluble and membrane proteins demonstrated that the large proteins induced under limitation by phosphorus and iron were associated with the membranes. However, small iron-repressible proteins were located in the soluble fraction. Isolation with anion-exchange chromatography and N-terminal sequencing of iron-repressible, 23-kDa Proteins from D. tertiolecta, P. tricornutum, and Chaetoceros gracilis revealed that these small soluble proteins have strong homology with the N-terminal sequence of flavodoxins from Azotobacter and Clostridium. The identity of the flavodoxin from D. tertiolecta was confirmed by immunodetection using antiflavodoxin raised against Chlorella. Flavodoxin was detected only under iron deprivation and was absent from nitrogen-and phosphorus-limited algae. Flavodoxin is a prime candidate for a molecular probe of iron limitation in the ocean. The requirements to confirm its utility in nature are discussed.     

Substrate-induced lipase gene expression and aflatoxin production in Aspergillus parasiticus and Aspergillus flavus

AIMS: To establish a relationship between lipase gene expression and aflatoxin production by cloning the lipA gene and studying its expression pattern in several aflatoxigenic and nontoxigenic isolates of Aspergillus flavus and A. parasiticus. METHODS AND RESULTS: We have cloned a gene, lipA, that encodes a lipase involved in the breakdown of lipids from aflatoxin-producing A. flavus, A. parasiticus and two nonaflatoxigenic A. flavus isolates, wool-1 and wool-2. The lipA gene was transcribed under diverse media conditions, however, no mature mRNA was detected unless the growth medium was supplemented with 0.5% soya bean or peanut oil or the fungus was grown in lipid-rich medium such as coconut medium. The expression of the lipase gene (mature mRNA) under substrate-induced conditions correlated well with aflatoxin production in aflatoxigenic species A. flavus (SRRC 1007) and A. parasiticus (SRRC 143). CONCLUSIONS: Substrate-induced lipase gene expression might be indirectly related to aflatoxin formation by providing the basic building block 'acetate' for aflatoxin synthesis. No direct relationship between lipid metabolism and aflatoxin production can be ascertained, however, lipase gene expression correlates well with aflatoxin formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Lipid substrate induces and promotes aflatoxin formation. It gives insight into genetic and biochemical aspects of aflatoxin formation.     

The lipA gene of Serratia marcescens which encodes an extracellular lipase having no N-terminal signal peptide.

The lipA gene encoding an extracellular lipase was cloned from the wild-type strain of Serratia marcescens Sr41. Nucleotide sequencing showed a major open reading frame encoding a 64.9-kDa protein of 613 amino acid residues; the deduced amino acid sequence contains a lipase consensus sequence, GXSXG. The lipase had 66 and 56% homologies with the lipases of Pseudomonas fluorescens B52 and P. fluorescens SIK W1, respectively, but did not show any overall homology with lipases from other origins. The Escherichia coli cells carrying the S. marcescens lipA gene did not secrete the lipase into the medium. The S. marcescens lipase had no conventional N-terminal signal sequence but was also not subjected to any processing at both the N-terminal and C-terminal regions. A specific short region similar to the regions of secretory proteins having no N-terminal signal peptide was observed in the amino acid sequence. Expression of the lipA gene in S. marcescens was affected by the carbon source and the addition of Tween 80.     

Genetic and biochemical characterization of a new extracellular lipase from Streptomyces cinnamomeus.

Streptomyces cinnamomeus Tü89 secretes a 30-kDa esterase and a 50-kDa lipase. The lipase-encoding gene, lipA, was cloned from genomic DNA into Streptomyces lividans TK23 with plasmid vector pIJ702. Two lipase-positive clones were identified; each recombinant plasmid had a 5.2-kb MboI insert that contained the complete lipA gene. The two plasmids differed in the orientation of the insert and the degree of lipolytic activity produced. The lipA gene was sequenced; lipA encodes a proprotein of 275 amino acids (29,213 Da) with a pI of 5.35. The LipA signal peptide is 30 amino acids long, and the mature lipase sequence is 245 amino acids long (26.2 kDa) and contains six cysteine residues. The conserved catalytic serine residue of LipA is in position 125. Sequence similarity of the mature lipases (29% identity, 60% similarity) was observed mainly in the N-terminal 104 amino acids with the group II Pseudomonas lipases; no similarity to the two Streptomyces lipase sequences was found. lipA was also expressed in Escherichia coli under the control of lacZ promoter. In the presence of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG), growth of the E. coli clone was severely affected, and the cells lysed in liquid medium. Lipase activity in the E. coli clone was found mainly in the pellet fraction. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, three additional protein bands of 50, 29, and 27 kDa were visible. The 27-kDa protein showed lipolytic activity and represents the mature lipase; the 29- and 50-kDa forms showed no activity and very probably represent the unprocessed form and a dimeric misfolded form, respectively. For higher expression of lipA in S. lividans, the gene was cloned next to the strong aphII promoter. In contrast to the lipA-expressing E. coli clone, S. cinnamomeus and the corresponding S. lividans clone secreted only an active protein of 50 kDa. The lipase showed highest activity with C6 and C18 triglycerides; no activity was observed with phospholipids, Tween 20, or p-nitrophenylesters. Upstream of lipA and in the same orientation, an open reading frame, orfA, is found whose deduced protein sequence (519 amino acids) shows similarity to various membrane-localized transporters. Downstream of lipA and in the opposite orientation, an open reading frame, orfB (encoding a 199-amino-acid protein) is found, which shows no conspicuous sequence similarity to known proteins, other than an NAD and flavin adenine dinucleotide binding-site sequence.     

溶藻弧菌铁载体合成及外膜蛋白表达的研究

初步研究了海洋动物病原菌溶藻弧菌的铁摄取机制。溶藻弧菌能够在高浓度铁螯合剂2-2二联吡啶的培养基中存活。在限铁环境中,溶藻弧菌生长受到抑制,补加铁可以消除这种抑制作用。通过铁载体定量检测,发现分离于发病鱼体的溶藻弧菌MVP01产铁载体量大于分离于海水的菌株No·1·1587。互补实验证明溶藻弧菌的铁载体粗提物能够被铁载体合成缺陷的大肠杆菌突变株AN93利用。在铁限制培养环境中,溶藻弧菌合成了约80kD铁调控外膜蛋白。铁摄取系统在溶藻弧菌的生存和致病性方面,都有重要的作用。     

Utilization of transferrin-bound iron by Haemophilus species of human and porcine origins

Haemophilus influenzae and H. haemolyticus acquired iron bound to human transferrin but not to human lactoferrin, ovo- or porcine transferrins. Conversely the swine pathogens H. pleuropneumoniae and H. parasuis used iron bound only to porcine transferrin. Growth under conditions of iron deprivation induced the production of siderophores and iron-repressible outer membrane proteins in H. parainfluenzae, H. paraphrophilus and H. parasuis but not in H. influenzae, H. haemolyticus or H. pleuropneumoniae. The latter 3 Haemophilus species appear to sequester transferrin bound iron via a siderophore-independent mechanism. However, the ability to produce iron chelating compounds did not enable H. parainfluenzae or H. paraphrophilus to utilize transferrin bound iron.