Iron Acquisition from Natural Organic Matter by an Aerobic Pseudomonas mendocina Bacterium: Siderophores and Cellular Iron Status

This research investigated the potential role of siderophores in aerobic microbial Fe acquisition from natural organic matter (NOM; XAD-8 isolate and reverse osmosis concentrate pre- and post-Chelex® treatment) through the use of a siderophore-producing Pseudomonas mendocina wild type (WT) bacterium and an engineered mutant (Mt) that was incapable of siderophore production. NOM had complex effects on microbial growth under Fe-limited conditions as measured by optical density, most likely because of the presence of other toxic (trace) metals such as Al, NOM binding interference with additional trace metal nutrients, and/or biofilm development. However, a bioassay for cellular Fe status showed that both WT and Mt readily acquired Fe naturally associated with NOM. Thus, while siderophores may be useful for Fe acquisition from NOM by P. mendocina, they do not appear to be essential for this process.     

Roles of Siderophores,Oxalate, and Ascorbate in Mobilization of Iron from Hematite by the Aerobic Bacterium Pseudomonas mendocina

In aerobic, circumneutral environments, the essential element Fe occurs primarily in scarcely soluble mineral forms. We examined the independent and combined effects of a siderophore, a reductant (ascorbate), and a low-molecular-weight carboxylic acid (oxalate) on acquisition of Fe from the mineral hematite (α-Fe₂O₃) by the obligate aerobe Pseudomonas mendocina ymp. A site-directed ΔpmhA mutant that was not capable of producing functional siderophores (i.e., siderophore⁻ phenotype) did not grow on hematite as the only Fe source. The concentration of an added exogenous siderophore (1 μM desferrioxamine B [DFO-B]) needed to restore wild-type (WT)-like growth kinetics to the siderophore⁻ strain was ∼50-fold less than the concentration of the siderophore secreted by the WT organism grown under the same conditions. The roles of a reductant (ascorbate) and a simple carboxylic acid (oxalate) in the Fe acquisition process were examined in the presence and absence of the siderophore. Addition of ascorbate (50 μM) alone restored the growth of the siderophore⁻ culture to the WT levels. A higher concentration of oxalate (100 μM) had little effect on the growth of a siderophore⁻ culture; however, addition of 0.1 μM DFO-B and 100 μM oxalate restored the growth of the mutant to WT levels when the oxalate was prereacted with the hematite, demonstrating that a metabolizing culture benefits from a synergistic effect of DFO-B and oxalate.Iron (Fe) is essential for almost all life. However, in aerobic, circumneutral environments, Fe is bound primarily in scarcely soluble minerals and amorphous solids [e.g., the solubility product (K_SP) for amorphous Fe(OH)₃ is 10⁻³⁸] (53) and is therefore poorly bioavailable. Aerobic microorganisms directly transform mineral-bound Fe(III) into soluble, highly bioavailable forms (1), overcoming significant kinetic and thermodynamic barriers to mineral dissolution and serving as primary transporters of Fe from the geosphere into global biogeochemical cycles.A primary means by which aerobic microorganisms enhance Fe mobility and bioavailability is by secreting siderophores, which are structurally diverse, low-molecular-weight chelating agents with extremely high affinities for Fe(III) (12, 27, 37, 40). Fe(III)-siderophore stability constants can be as high as 10⁵² (1, 40), which is many orders of magnitude higher than the stability constants for low-molecular-weight organic acids, such as oxalic acid [for Fe(III) + 3 oxalate ⇆ Fe(oxalate)₃, K = 10^18.6] (45). While their high affinity for Fe(III) is clearly important for helping siderophores mobilize Fe from Fe(III) (hydr)oxides in the aqueous phase, the mechanisms of Fe mobilization appear to be complex and are the subject of much recent study (14, 17, 18, 26, 28, 49). In particular, the role of siderophores in ligand-promoted dissolution mechanisms has undergone careful evaluation in vitro. The model is described simply here as follows for amorphous Fe(OH)₃ and has been described in detail by Kraemer (26): Fe(OH)₃ + 3H⁺ ⇆ Fe(III) + 3H₂O (K_SP) (equation 1); Fe(III) + H₃L ⇆ FeL + 3H⁺ (K_FeL) (equation 2); and Fe(OH)₃ + H₃L ⇆ FeL + 3H₂O (K_eq = K_SPK_FeL = [FeL]/[H₃L]) (equation 3). The concentration of the solubilized FeL complex, according to equation 3, is determined as follows: [FeL] = [H₃L]K_{SP ×} K_FeL. The estimated concentration of siderophores in carbonic soil (∼10⁻⁸ to 10⁻⁷ M), combined with their strong affinities for Fe(III) (39), suggests that [FeL] could in principle easily be micromolar or higher and could support vigorous bacterial growth. However, the trishydroxamate siderophores that have been studied most to date adsorb only weakly to Fe(III) (hydr)oxide minerals, likely due to steric constraints, although charge repulsion may also play a role for positively charged siderophores, such as desferrioxamine B (DFO-B) (6, 26, 41, 42). Therefore, it has been proposed that siderophores act primarily in conjunction with other molecules, such as simple plant-derived carboxylic acids or reductants, which interact more strongly with mineral surfaces and release Fe directly through ligand-promoted and/or reductive mechanisms (52). This proposed “synergistic effect,” in which the combined effect of various elements is greater than the sum of the individual effects, suggests that an interaction of biogenic molecules may overcome kinetic and thermodynamic barriers to the release of Fe from minerals in the presence of siderophores. The role of the siderophore in such a synergistic system is not a direct role in surface processes; rather, the siderophore maintains a low concentration of aqueous Fe in equilibrium with the mineral (an Fe sink), thus driving the reaction toward more dissolution (26, 41). Only a low concentration of a siderophore relative to the concentrations of surface-reacting organic species is required to promote efficient dissolution (26).The synergistic effect has been observed directly in in vitro, abiotic experiments using combinations of microbe-derived siderophores and simple organic acids. A combination of environmentally relevant concentrations of oxalate (1 to 80 μM) and DFO-B (40 μM), for example, doubled the rate of Fe(III) hydroxide mineral (goethite) dissolution compared with the rate when only oxalate or DFO-B was present in a recent in vitro study (6). Actively metabolizing aerobic bacteria, which can move Fe from solution into cells and recycle or release new siderophores back into the medium, might be expected to promote the synergistic siderophore-carboxylic acid interaction even further in a batch system. Likewise, it has been suggested that organic reductants may work synergistically with siderophores. In particular, a recent study showed that exogenously added reductants significantly enhance the bioavailability of Fe to an aerobic siderophore-producing bacterium, Pseudomonas mendocina ymp (15), isolated from the Nevada Test Site and used in the work described here.As an obligate aerobe, P. mendocina ymp does not have dissimilatory reduction pathways, so that its use of iron (hydr)oxide minerals is only for acquisition of nutritional Fe and not for cellular respiration. In contrast to dissimilatory Fe-reducing bacteria, which require millimolar concentrations of Fe (2, 29-31, 36, 43, 50), P. mendocina ymp requires micromolar concentrations (19, 20, 24, 32-34). Previously, this strain''s ability to dissolve and use various mineral forms of Fe was quantified in a series of microbial growth studies (23, 24, 32-34). P. mendocina ymp is known to produce hydroxamate-containing siderophores that increase the rate of dissolution of the Fe oxide mineral hematite. A recent study demonstrated that reductants significantly enhanced the bioavailability of Fe-(hydr)oxide minerals to P. mendocina (15). The ymp strain was also shown to have endogenous Fe(III)-reducing activity, which Hersman et al. suggested could be involved in solubilizing ferric minerals (24). Likewise, closely related strains of Pseudomonas stutzeri have been shown to produce pyridine-2,6-bis(thiocarboxylic acid) (PDTC), which they can use in the reduction, transport, and detoxification of metals and metalloids (11, 16, 51). However, control experiments showed that P. mendocina did not secrete molecules that exhibited a significant amount of reducing activity under the conditions used in this study (see the supplemental material). Notably, this species does not appear to contain a set of PDTC biosynthesis genes.In this work we used the wild-type (WT) strain P. mendocina ymp along with a mutant with a site-directed markerless mutation that was not capable of producing siderophores (ΔpmhA mutant with the siderophore⁻ phenotype) (3) in a series of experiments examining siderophore use and potential synergistic effects with either a simple carboxylic acid (oxalate) or an exogenous reductant (ascorbate). Both ascorbate and oxalate are plant products that are frequently found in the shallow subsurface; their effects on in vitro Fe (hydr)oxide dissolution have been well described (6).     

Analysis and comparison of a set of expressed sequence tags of the parthenogenetic water flea <Emphasis Type="Italic">Daphnia carinata</Emphasis>

The water flea Daphnia carinata (D. carinata) reproduces both sexually and parthenogenetically, yet little is known about the genes involved in these processes. To further clarify the reproductive biology of Daphnia and elucidate their unique mechanism of reproductive transformation, we have generated and characterized an expressed sequence tag (EST) data set from D. carinata. A set of 1,495 clusters were generated from sequencing 3,072 randomly chosen clones from a parthenogenetic, juvenile water flea cDNA library. The nucleic acid and deduced amino acid sequences were compared with known GenBank sequences. Functional annotation found that 959 clusters showed significant homology with known genes involved in a broad range of activities, including metabolism, translation, development and reproduction, as well as genes involved in sensing environmental factors. We speculate that genes involved in development and reproduction, along with genes that allow the organism to sense changes in the environment, play important roles in the process of parthenogenetic reproduction and could be markers of the early steps of sexual differentiation. Additionally, 86% of the D. Carinata unique sequences could be stringently mapped to the D. pulex genome, of which 125 mapped to intergenic and intronic regions on the current assembly. Our results provide practical insight into crustacean reproductive biology, in addition to establishing a new animal model for reproductive and developmental biology. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Xiaoqian Xu and Shuhui Song contributed equally to this work. Nucleotide sequence data reported are available in the GenBank databases under the accession numbers: GD269049−GD272045.     

Stetteria hydrogenophila, gen. nov. and sp. nov., a novel mixotrophic sulfur-dependent crenarchaeote isolated from Milos, Greece

A new hyperthermophilic, strictly anaerobic crenarchaeote, Stetteria hydrogenophila DSM11227 representing a new genus within the family of Desulfurococcaceae, was isolated from the sediment of a marine hydrothermal system at Paleohori Bay in Milos, Greece. Cells are gram-negative irregular and disc-shaped cocci, 0.5–1.5 μm in diameter, which are flagellate and can form cytoplasmatic protrusions up to 2 μm in length. The strain grew optimally at 95°C at pH 6.0 and at a NaCl concentration of 3%. The organism grew mixotrophically on peptide substrates. It required elemental sulfur as an external electron acceptor, and in addition, its growth was completely dependent on the presence of molecular hydrogen. Sulfur could be replaced by thiosulfate. H₂S, CO₂, acetate, and ethanol were identified as products of metabolism. The G + C content of DNA was 65 mol%. Analysis of its phylogenetic position by sequence analysis of 16S rRNA placed this organism in the family of Desulfurococcaceae. The dependence of this organism on both hydrogen and sulfur during growth on peptide substrates distinguishes Stetteria from all previously described species of Crenarchaeota. Received: September 4, 1996 / Accepted: November 12, 1996     

Genomic insights into the iron uptake mechanisms of the biomining microorganism Acidithiobacillus ferrooxidans

Commercial bioleaching of copper and the biooxidation of gold is a cost-effective and environmentally friendly process for metal recovery. A partial genome sequence of the acidophilic, bioleaching bacterium Acidithiobacillus ferrooxidans is available from two public sources. This information has been used to build preliminary models that describe how this microorganism confronts unusually high iron loads in the extremely acidic conditions (pH 2) found in natural environments and in bioleaching operations. A. ferrooxidans contains candidate genes for iron uptake, sensing, storage, and regulation of iron homeostasis. Predicted proteins exhibit significant amino acid similarity with known proteins from neutrophilic organisms, including conservation of functional motifs, permitting their identification by bioinformatics tools and allowing the recognition of common themes in iron transport across distantly related species. However, significant differences in amino acid sequence were detected in pertinent domains that suggest ways in which the periplasmic and outer membrane proteins of A. ferrooxidans maintain structural integrity and relevant protein-protein contacts at low pH. Unexpectedly, the microorganism also contains candidate genes, organized in operon-like structures that potentially encode at least 11 siderophore systems for the uptake of Fe(III), although it does not exhibit genes that could encode the biosynthesis of the siderophores themselves. The presence of multiple Fe(III) uptake systems suggests that A. ferrooxidans can inhabit aerobic environments where iron is scarce and where siderophore producers are present. It may also help to explain why it cannot tolerate high Fe(III) concentrations in bioleaching operations where it is out-competed by Leptospirillum species.

     

Growth of Pseudomonas mendocina on Fe(III) (Hydr)Oxides

Although iron (Fe) is an essential element for almost all living organisms, little is known regarding its acquisition from the insoluble Fe(III) (hydr)oxides in aerobic environments. In this study a strict aerobe, Pseudomonas mendocina, was grown in batch culture with hematite, goethite, or ferrihydrite as a source of Fe. P. mendocina obtained Fe from these minerals in the following order: goethite > hematite > ferrihydrite. Furthermore, Fe release from each of the minerals appears to have occurred in excess, as evidenced by the growth of P. mendocina in the medium above that of the insoluble Fe(III) (hydr)oxide aggregates, and this release was independent of the mineral's surface area. These results demonstrate that an aerobic microorganism was able to obtain Fe for growth from several insoluble Fe minerals and did so with various growth rates.     

Ferrichrome in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> – an iron transport and iron storage compound

Schizosaccharomyces pombe has been assumed not to produce siderophores. Nevertheless, the genomic sequence of this fission yeast revealed the presence of siderophore biosynthetic genes for hydroxamates. Applying a bioassay based on an Aspergillus nidulans strain deficient in siderophore biosynthesis, and using reversed-phase HPLC and mass spectrometry analysis, we demonstrate that S. pombe excretes and accumulates intracellularly the hydroxamate-type siderophore ferrichrome. Under iron-limiting conditions, the cellular ferrichrome pool was present in the desferri-form, while under iron-rich conditions, in the ferri-form. In contrast to S. pombe, hydroxamate-type siderophores could not be detected in two other yeast species, Saccharomyces cerevisiae and Candida albicans.     

Genomic analysis and secondary metabolite production in Bacillus amyloliquefaciens AS 43.3: A biocontrol antagonist of Fusarium head blight

The complete genome of the biocontrol antagonist Bacillus amyloliquefaciens AS 43.3 is reported. B. amyloliquefaciens AS 43.3 has previously been shown to be effective in reducing Fusarium head blight in wheat. The 3.9 Mbp genome was sequenced, assembled, and annotated. Genomic analysis of the strain identified 9 biosynthetic gene clusters encoding secondary metabolites associated with biocontrol activity. The analysis identified five non-ribosomal peptide synthetase clusters encoding three lipopeptides (surfactin, iturin, and fengycin), a siderophore (bacillibactin), and the antibiotic dipeptide bacilysin. In addition, three polyketide synthetase clusters were identified which encoded for the antibacterials: bacillaene, difficidin, and macrolactin. In addition to the non-ribosomal mediated biosynthetic clusters discovered, we identified a ribosomally encoded biosynthetic cluster that produces the antibiotic plantazolicin. To confirm the gene clusters were functional, cell-free culture supernatant was analyzed using LC–MS/MS. The technique confirmed the presence of all nine metabolites or their derivatives. The study suggests the strain is most likely a member of the B. amyloliquefaciens subsp. plantarium clade. Comparative genomics of eight completed genomes of B. amyloliquefaciens identify the core and pan-genomes for the species, including identifying genes unique to the biocontrol strains. This study demonstrates the growing importance of applying genomic-based studies to biocontrol organisms of plant pathogens which can enable the rapid identification of bioactive metabolites produced by a prospective biological control organism. In addition, this work provides a foundation for a mechanistic understanding of the B. amyloliquefaciens AS 43.3/Fusarium head blight biocontrol interaction.     

Accumulation of polyhydroxyalkanoates by Microlunatus phosphovorus under various growth conditions

Microlunatus phosphovorus is an activated-sludge bacterium with high levels of phosphorus-accumulating activity and phosphate uptake and release activities. Thus, it is an interesting model organism to study biological phosphorus removal. However, there are no studies demonstrating the polyhydroxyalkanoate (PHA) storage capability of M. phosphovorus, which is surprising for a polyphosphate-accumulating organism. This study investigates in detail the PHA storage behavior of M. phosphovorus under different growth conditions and using different carbon sources. Pure culture studies in batch-growth systems were conducted in shake-flasks and in a bioreactor, using chemically defined growth media with glucose as the sole carbon source. A batch-growth system with anaerobic–aerobic cycles and varying concentrations of glucose or acetate as the sole carbon source, similar to enhanced biological phosphorus removal processes, was also employed. The results of this study demonstrate for the first time that M. phosphovorus produces significant amounts of PHAs under various growth conditions and with different carbon sources. When the PHA productions of all cultivations were compared, poly(3-hydroxybutyrate) (PHB), the major PHA polymer, was produced at about 20–30% of the cellular dry weight. The highest PHB production was observed as 1,421 mg/l in batch-growth systems with anaerobic–aerobic cycles and at 4 g/l initial glucose concentration. In light of these key results regarding the growth physiology and PHA-production capability of M. phosphovorus, it can be concluded that this organism could be a good candidate for microbial PHA production because of its advantages of easy growth, high biomass and PHB yield on substrate and no significant production of fermentative byproducts.     

Genome Sequence of Pseudomonas mendocina DLHK,Isolated from a Biotrickling Reactor

Pseudomonas mendocina DLHK is an aerobic bacterium isolated from a biotrickling reactor which can remove nitric oxide, a common air pollutant from combustion exhaust gas. Here, we present the draft genome of Pseudomonas mendocina DLHK.     

Sequence Analysis of a 34.7-kb DNA Segment from the Genome of Buchnera aphidicola (Endosymbiont of Aphids) Containing groEL, dnaA, the atp operon, gidA, and rho

Buchnera aphidicola is a prokaryotic endosymbiont of the aphid Schizaphis graminum. From past and present nucleotide sequence analyses of the B. aphidicola genome, we have assembled a 34.7-kilobase (kb) DNA segment. This segment contains genes coding for 32 open reading frames (ORFs), which corresponded to 89.9% of the DNA. All of these ORFs could be identified with homologous regions of the Escherichia coli genome. The order of the genes with established functions was groELS–trmE–rnpA–rpmH–dnaA–dnaN–gyrB–atpCDGAHFEB–gidA–fdx–hscA– hscB–nifS–ilvDC–rep–trxA–rho. The order of genes in small DNA fragments was conserved in both B. aphidicola and E. coli. Most of these fragments were in approximately the same region of the E. coli genome. The latter organism, however, contained many additional inserted genes within and between the fragments. The results of the B. aphidicola genome analyses indicate that the endosymbiont has many properties of free-living bacteria. Received: 15 August 1997 / Accepted: 29 August 1997     

Microbial biomass and productivity in seagrass beds

This research focused on whether bacteria living in aerobic environments where Fe is often a limiting nutrient could access Fe associated with the clay mineral kaolinite. Kaolinite is one of the most abundant clays at the Earth's surface, and it often contains trace quantities of Fe as surface precipitates, accessory minerals, and structural substitutions. We hypothesized that aerobic bacteria may enhance kaolinite dissolution as a means of obtaining associated Fe. To test this hypothesis, we conducted microbial growth experiments in the presence of an aerobic Pseudomonas mendocina     

Characterization of the pyoverdines ofAzotobacter vinelandii ATCC 12 837 with regard to heterogeneity

Summary Azotobacter vinelandii strain ATCC 12 837 produces peptide siderophores of the general class known as pyoverdines. In the past, it was assumed that a single well-defined pyoverdine was produced by each parent microorganism. However, there are a number of reports of incompletely characterized pyoverdines that demonstrate heterogeneity in pyoverdine preparations obtained from a single organism, but the nature of this phenomena has not been explained. This study shows thatA. vinelandii does indeed produce more than one pyoverdine and that these compounds differ in their peptide components. The metabolism of these siderophores suggests that only one of them is a true siderophore while the others are metabolic byproducts. It was demonstrated that this phenomenon is likely due to intrinsic limitations of the synthetase complex involved in the biosynthesis of these compounds. Characterization of two of the major pyoverdines produced demonstrated that they are novel compounds, although they belonged to theAzotobacter-type family of pyoverdines.     

Genome-based cryptic gene discovery and functional identification of NRPS siderophore peptide in Streptomyces peucetius

Identification of secondary metabolites produced by cryptic gene in bacteria may be difficult, but in the case of nonribosomal peptide (NRP)-type secondary metabolites, this study can be facilitated by bioinformatic analysis of the biosynthetic gene cluster and tandem mass spectrometry analysis. To illustrate this concept, we used mass spectrometry-guided bioinformatic analysis of genomic sequences to identify an NRP-type secondary metabolite from Streptomyces peucetius ATCC 27952. Five putative NRPS biosynthetic gene clusters were identified in the S. peucetius genome by DNA sequence analysis. Of these, the sp970 gene cluster encoded a complete NRPS domain structure, viz., C-A-T-C-A-T-E-C-A-T-C-A-T-C domains. Tandem mass spectrometry revealed that the functional siderophore peptide produced by this cluster had a molecular weight of 644.4 Da. Further analysis demonstrated that the siderophore peptide has a cyclic structure and an amino acid composition of AchfOrn–Arg–hOrn–hfOrn. The discovery of functional cryptic genes by analysis of the secretome, especially of NRP-type secondary metabolites, using mass spectrometry together with genome mining may contribute significantly to the development of pharmaceuticals such as hybrid antibiotics.     

Evolution of Growth Hormone in Primates: The GH Gene Clusters of the New World Monkeys Marmoset (Callithrix jacchus) and White-Fronted Capuchin (Cebus albifrons)

The GH gene cluster in marmoset, Callithrix jacchus, comprises eight GH-like genes and pseudogenes and appears to have arisen as a consequence of gene duplications occurring independently of those leading to the human GH gene cluster. We report here the complete sequence of the marmoset GH gene locus, including the intergenic regions and 5′ and 3′ flanking sequence, and a study of the multiple GH-like genes of an additional New World monkey (NWM), the white-fronted capuchin, Cebus albifrons. The marmoset sequence includes 945 nucleotides (nt) of 5′ flanking sequence and 1596 nt of 3′ flanking sequence that are “unique”; between these are eight repeat units, including the eight GH genes/pseudogenes. The breakpoints between these repeats are very similar, indicating a regular pattern of gene duplication. These breakpoints do not correspond to those found in the much less regular human GH gene cluster. This and phylogenetic analysis of the repeat units within the marmoset gene cluster strongly support the independent origin of these gene clusters, and the idea that the episode of rapid evolution that occurred during GH evolution in primates preceded the gene duplications. The marmoset GH gene cluster also differs from that of human in having fewer and more evenly distributed Alu sequences (a single pair in each repeat unit) and a “P-element” upstream of every gene/pseudogene. In human there is no P-element upstream of the gene encoding pituitary GH, and these elements have been implicated in placental expression of the other genes of the cluster. The GH gene clusters in marmoset and capuchin appear to have arisen as the consequence of a single-gene duplication event, but in capuchin there was then a remarkable expansion of the GH locus, giving at least 40 GH-like genes and pseudogenes. Thus even among NWMs the GH gene cluster is very variable. [Reviewing Editor: Nicolas Galtier]     

Hydroxamate-siderophore production and utilization by marine eubacteria

Siderophore (iron-binding chelator) production was examined in 30 strains of open ocean bacteria from the generaVibrio, Alteromonas, Alcaligenes, Pseudomonas, andPhotobacterium. The results showed that hydroxamate-type siderophore production was widely distributed in various marine species, except for isolates ofAlteromonas macleodii andV. nereis. In all cases, the ability to produce siderophores was under the control of iron levels in the medium and satisfied the iron requirements of the siderophore bioassay organism. On the basis of chemical assay and bacterial bioassays, none of the examined isolates produced phenolate-type siderophores. Several isolates produces siderophores that were neither hydroxamatenor phenolate-type siderophores. Some strains such asAlteromonas communis produce siderophores that could be used by many other isolates. In contrast, the siderophore produced byAlcaligenes venustus had little cross-strain utilization. These findings suggest that the ability to produce siderophores may be common to open ocean bacteria.     

Bioproducts from <Emphasis Type="Italic">Aureobasidium pullulans,</Emphasis> a biotechnologically important yeast

It has been well documented that Aureobasidium pullulans is widely distributed in different environments. Different strains of A. pullulans can produce amylase, proteinase, lipase, cellulase, xylanase, mannanase, transferases, pullulan, siderophore, and single-cell protein, and the genes encoding proteinase, lipase, cellulase, xylanase, and siderophore have been cloned and characterized. Therefore, like Aspergillus spp., it is a biotechnologically important yeast that can be used in different fields. So it is very important to sequence the whole genomic DNA of the yeast cells in order to find new more bioproducts and novel genes from this yeast.     

Evidence for an ergot alkaloid gene cluster in Claviceps purpurea

A gene (cpd1) coding for the dimethylallyltryptophan synthase (DMATS) that catalyzes the first specific step in the biosynthesis of ergot alkaloids, was cloned from a strain of Claviceps purpurea that produces alkaloids in axenic culture. The derived gene product (CPD1) shows only 70% similarity to the corresponding gene previously isolated from Claviceps strain ATCC 26245, which is likely to be an isolate of C. fusiformis. Therefore, the related cpd1 most probably represents the first C. purpurea gene coding for an enzymatic step of the alkaloid biosynthetic pathway to be cloned. Analysis of the 3′-flanking region of cpd1 revealed a second, closely linked ergot alkaloid biosynthetic gene named cpps1, which codes for a 356-kDa polypeptide showing significant similiarity to fungal modular peptide synthetases. The protein contains three amino acid-activating modules, and in the second module a sequence is found which matches that of an internal peptide (17 amino acids in length) obtained from a tryptic digest of lysergyl peptide synthetase 1 (LPS1) of C. purpurea, thus confirming that cpps1 encodes LPS1. LPS1 activates the three amino acids of the peptide portion of ergot peptide alkaloids during D-lysergyl peptide assembly. Chromosome walking revealed the presence of additional genes upstream of cpd1 which are probably also involved in ergot alkaloid biosynthesis: cpox1 probably codes for an FAD-dependent oxidoreductase (which could represent the chanoclavine cyclase), and a second putative oxido-reductase gene, cpox2, is closely linked to it in inverse orientation. RT-PCR experiments confirm that all four genes are expressed under conditions of peptide alkaloid biosynthesis. These results strongly suggest that at least some genes of ergot alkaloid biosynthesis in C. purpurea are clustered, opening the way for a detailed molecular genetic analysis of the pathway. Received: 26 August 1998 / Accepted: 19 October 1998     

The purple non‐sulfur bacterium Rhodopseudomonas palustris produces novel petrobactin‐related siderophores under aerobic and anaerobic conditions

Many bacteria produce siderophores to bind and take up Fe(III), an essential trace metal with extremely low solubility in oxygenated environments at circumneutral pH. The purple non‐sulfur bacterium Rhodopseudomonas palustris str. CGA009 is a metabolically versatile model organism with high iron requirements that is able to grow under aerobic and anaerobic conditions. Siderophore biosynthesis has been predicted by genomic analysis, however, siderophore structures were not identified. Here, we elucidate the structure of two novel siderophores from R. palustris: rhodopetrobactin A and B. Rhodopetrobactins are structural analogues of the known siderophore petrobactin in which the Fe chelating moieties are conserved, including two 3,4‐dihydroxybenzoate and a citrate substructure. In the place of two spermidine linker groups in petrobactin, rhodopetrobactins contain two 4,4′‐diaminodibutylamine groups of which one or both are acetylated at the central amine. We analyse siderophore production under different growth modes and show that rhodopetrobactins are produced in response to Fe limitation under aerobic as well as under anaerobic conditions. Evaluation of the chemical characteristics of rhodopetrobactins indicates that they are well suited to support Fe acquisition under variable oxygen and light conditions.