The unique role of siderophore in marine-derived Aureobasidium pullulans HN6.2

The l-ornithine-N ⁵-monooxygenase structural gene (SidA gene, accession number: FJ769160) was isolated from both the genomic DNA and cDNA of the marine yeast Aureobasidium pullulans HN6.2 by inverse PCR and RT-PCR. An open reading frame of 1,461 bp encoding a 486 amino acid protein (isoelectric point: 7.79) with calculated molecular weight of 55.4 kDa was characterized. The promoter of the gene (intronless) was located from −1 to −824 and had three HGATAR boxes which were putative binding motifs for the respective DNA-binding motifs and one CATA box. The SidA gene in A. pullulans HN6.2 was disrupted by integrating the hygromycin B phosphotransferase (HPT) gene into Open Reading Frame of the SidA gene using homologous recombination. Of all the disruptants obtained, one strain S6 (∆sidA) did not synthesize both intracellular and extracellular fusigen so that it could not inhibit growth of the pathogenic bacteria Vibrio anguillarum and Vibrio parahaemolyticus. The disruptant S6 did not grow in the iron-deplete medium and seawater medium because cell budding was stopped, but could grow in the iron-replete medium with 10 μM Fe³⁺ and Fe²⁺. H₂O₂ in the medium was more toxic to the disruptant S6 than to its wild type HN6.2. Thus, we infer that the fusigen produced by the marine-derived A. pullulans HN6.2 can play a unique role in chelating, uptake and concentration of iron to maintain certain proper physiological functions within the cells and secretion of siderophore may represent an efficient tool to eliminate competitors to compete for limiting nutritional resources in marine environments.     

Siderophore production by the marine-derived Aureobasidium pullulans and its antimicrobial activity

Over 300 yeast strains isolated from different marine environments were screened for their ability to produce siderophore. Among them, only the yeast strain HN6.2 which was identified to be Aureobasidium pullulans was found to produce high level of the siderophore. Under the optimal conditions, this yeast strain could produce 1.1 mg/ml of the siderophore. The crude siderophore produced by the yeast strain HN6.2 was able to inhibit cell growth of Vibrio anguillarum and Vibrio parahaemolyticus, isolated from the diseased marine animals.     

Chemical and biological characterization of siderophore produced by the marine-derived Aureobasidium pullulans HN6.2 and its antibacterial activity

After analysis using HPLC and electronic ion spray mass spectroscopy, the purified siderophore produced by the marine-derived Aureobasidium pullulans HN6.2 was found to be fusigen. The purified desferric fusigen still had strong inhibition of growth of the pathogenic Vibrio anguillarum while the fusigen chelated by Fe³⁺ lost the ability to inhibit the growth of the pathogenic bacterium. The added iron in the medium repressed expression of the hydroxylase gene encoding ornithine N⁵-oxygenase that catalyzes the N⁵-hydroxylation of ornithine for the first step of siderophore biosynthesis in the yeast cells while expression of the hydroxylase gene in the yeast cells grown in the medium plus ornithine was enhanced.     

A comparative study of siderophore production by fungi from marine and terrestrial habitats

Siderophore producing potential of 20 fungal isolates (same 10 species from each marine and terrestrial habitat) were examined and compared. Except marine Aspergillus flavus, all isolates produced siderophores as evidenced by positive reaction in FeCl₃ test, CAS assay and CAS agar plate test. The results indicated widespread occurrence of siderophores in both the habitats. Examination of the chemical nature of siderophores revealed that mucoraceous fungi produced carboxylate, while others produced hydroxamate siderophores. Thus, the nature of siderophore was found to be independent of habitat. Among all the isolates, Cunninghamella elegans (marine form) was maximum siderophore producer (1987.5 μg/ml) followed by terrestrial form of C. elegans (1248.75 μg/ml). There was no marked variation in siderophore concentration of Penicillium funiculosum strains. Comparison of quantification of siderophore production between marine and terrestrial revealed that four terrestrial isolates (Aspergillus niger, Aspergillus ochraceous, Penicillium chrysogenum, Penicillium citrinum) were ahead in siderophore production, while, the other four marine isolates (Aspergillus versicolor, C. elegans, Rhizopus sp., Syncephalastrum racemosum) were found to be more potent siderophore producers, indicating that they were equally competent.     

Downstream processing and characterization of pullulan from a novel colour variant strain of Aureobasidium pullulans FB-1

Exopolysaccharide produced by a new novel colour variant strain of Aureobasidium pullulans FB-1 was purified by cell harvesting and precipitation of the polymer. Various organic solvents were screened for pullulan precipitation. Isolation and purification of pullulan from fermentation broth was carried out using single-step purification strategy by isopropyl alcohol precipitation. Ratio of culture supernatant to isopropyl alcohol and time of precipitation were optimized for pullulan precipitation. Maximum yield (4.47%, w/v) of polysaccharide was obtained when two volumes of ice-cold isopropyl alcohol were added to one volume of supernatant with precipitation time of 12 h. IR spectra as well as carbon-13 and proton NMR spectra in aqueous solution of intact polysaccharide obtained from A. pullulans FB-1 and commercially available pullulan (Sigma, USA) revealed solely α-(1 → 6) linked maltosyl units, in accord with the generally accepted structure of pullulan. Maximum hydrolysis (94.25%) of purified pullulan at 50 °C by pullulanase was achieved under agitation (150 rpm) after 360 min.     

High pullulan yield is related to low UDP-glucose level and high pullulan-related synthases activity inAureobasidium pullulans Y68

Effects of different pH and carbon sources on pullulan production, UDP-glucose level and pullulan-related synthases activity inAureobasidium pullulans Y68 were examined. It was found that more pullulan was produced when the yeast strain was grown in the medium with initial pH 7.0 than when it was grown in the same medium with constant pH 6.0. The results also show that higher pullulan yield was obtained when the cells were grown in the medium containing glucose than when they were cultivated in the medium supplementing other carbon sources. Our results demonstrate that the more pullulan was synthesized, the less UDP-glucose was left in the cells ofA. pullulans Y68. However, it was observed that more pullulan was synthesized; the cells had higher pullulan-related synthase activity. Therefore, high pullulan yield was related to low UDP-glucose level and high pullulan-related synthases activity inAureobasidium pullulans Y68.     

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.     

Influence of different sugars on pullulan production and activities of α-phosphoglucose mutase, UDPG-pyrophosphorylase and glucosyltransferase involved in pullulan synthesis in Aureobasidium pullulans Y68

Effects of different sugars on pullulan production, UDP-glucose level, and activities of α-phosphoglucose mutase, UDPG-pyrophosphorylase and glucosyltransferase in Aureobasidium pullulans Y68 were examined. It was found that more pullulan was produced when the yeast strain was grown in the medium containing glucose than when it was cultivated in the medium supplementing other sugars. Our results demonstrate that when more pullulan was synthesized, less UDP-glucose was left in the cells of A. pullulans Y68. However, it was observed that more pullulan was synthesized, the cells had higher activities of α-phosphoglucose mutase, UDPG-pyrophosphorylase and glycosyltransferase. Therefore, high pullulan yield is related to high activities of α-phosphoglucose mutase, UDPG-pyrophosphorylase and glucosyltransferase in A. pullulans Y68 grown on different sugars. A pathway of pullulan biosynthesis in A. pullulan Y68 was proposed based on the results of this study and those from other researchers. This study will be helpful to metabolism-engineer the yeast strain to further enhance pullulan yield.     

Petrobactin,a siderophore produced by Alteromonas,mediates community iron acquisition in the global ocean

It is now widely accepted that siderophores play a role in marine iron biogeochemical cycling. However, the mechanisms by which siderophores affect the availability of iron from specific sources and the resulting significance of these processes on iron biogeochemical cycling as a whole have remained largely untested. In this study, we develop a model system for testing the effects of siderophore production on iron bioavailability using the marine copiotroph Alteromonas macleodii ATCC 27126. Through the generation of the knockout cell line ΔasbB::km^r, which lacks siderophore biosynthetic capabilities, we demonstrate that the production of the siderophore petrobactin enables the acquisition of iron from mineral sources and weaker iron-ligand complexes. Notably, the utilization of lithogenic iron, such as that from atmospheric dust, indicates a significant role for siderophores in the incorporation of new iron into marine systems. We have also detected petrobactin, a photoreactive siderophore, directly from seawater in the mid-latitudes of the North Pacific and have identified the biosynthetic pathway for petrobactin in bacterial metagenome-assembled genomes widely distributed across the global ocean. Together, these results improve our mechanistic understanding of the role of siderophore production in iron biogeochemical cycling in the marine environment wherein iron speciation, bioavailability, and residence time can be directly influenced by microbial activities.Subject terms: Biogeochemistry, Marine microbiology     

The current status of <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> in biotechnology

Different strains of the saprophytic yeast-like fungus Aureobasidium pullulans (Ascomycota: Dothideales) exhibit different biochemical characteristics, while their ubiquitous occurrence across diverse habitats and environmental conditions makes them an easily accessible source for biotechnological exploitation. They are useful in agricultural and industrial applications. Their antagonistic activities against postharvest pathogens make them suitable bioagents for the postharvest preservation of fruits and vegetables, while they possess antimicrobial activities against bacteria and fungi. Additionally, A. pullulans appears to be a potent source of single-cell protein. Many strains of A. pullulans harbor a wide range of industrially important enzymes, while the trademark exopolysaccharide pullulan that they produce has been extensively studied and is currently used in many applications. They also produce poly (β-l-malic acid), heavy oil liamocins, siderophore, and aubasidan-like β-glucan which are of interest for future applications. Ongoing studies suggest that A. pullulans holds many more interesting properties capable of further potential biotechnological applications.     

Fe(III)-complexes of the tripodal trishydroxamate siderophore basidiochrome: potential biological implications

One method of mobilization of iron by mycorrhizal organisms is through the secretion of small organic chelators called siderophores. Hydroxamate donor chelators are a common type of siderophore that is frequently used by fungal organisms. The primary siderophore that is produced by fungi from the genera Ceratobasidium and Rhizoctonia is the tripodal trishydroxamate siderophore basidiochrome. To gain some insight into the iron uptake mechanisms of these symbiotic fungi, the iron binding characteristics of basidiochrome were determined. It was found that basidiochrome exhibits a log β₁₁₀ of 27.8 ± 0.1 and a pFe value of 25.0. These values are similar to those of another fungal trishydroxamate siderophore, ferrichrome. The similarity in iron affinity between the two siderophores suggests that the structure of the backbone has little influence in complex formation due to the length of the pendant arms, although the identity of the terminating groups of the pendant arms is likely related to complex stability. The role of basidiochrome in the biogeochemical cycling of iron is also discussed.     

Effect of two-stage temperature on pullulan production by Aureobasidium pullulans

The effect of a two-stage cultivation temperature on the production of pullulan synthesized by Aureobasidium pullulans CGMCC1234 was investigated. Pullulan production was affected by temperature; although the optimum temperature for pullulan production was 26°C, the optimal temperature for cell growth was 32°C. Maximum pullulan production was achieved by growing A. pullulans in a first stage of 32°C for 2 days, and then in a second stage of 26°C for 2 days. Pullulan production using these two-stage temperatures significantly increased: about 27.80% (w/w) compared to constant-temperature fermentation (26°C for 4 days). The morphology of the A. pullulans (CGMCC 1234) was also affected by temperature; the lower temperature (26°C) supported unicellular biomass growth. Results of this study indicate that fermentation using two temperature stages is a promising method for pullulan production.     

Linear fusigen as the major hydroxamate siderophore of the ectomycorrhizal Basidiomycota Laccaria laccata and Laccaria bicolor

A screening for siderophores produced by the ectomycorrhizal fungi Laccaria laccata and Laccaria bicolor in synthetic low iron medium revealed the release of several different hydroxamate siderophores of which four major siderophores could be identified by high resolution mass spectrometry. While ferricrocin, coprogen and triacetylfusarinine C were assigned as well as other known fungal siderophores, a major peak of the siderophore mixture revealed an average molecular mass of 797 for the iron-loaded compound. High resolution mass spectrometry indicated an absolute mass of m/z = 798.30973 ([M + H]⁺). With a relative error of Δ = 0.56 ppm this corresponds to linear fusigen (C₃₃H₅₂N₆O₁₃Fe; MW = 797.3). The production of large amounts of linear fusigen by these basidiomycetous mycorrhizal fungi may possibly explain the observed suppression of plant pathogenic Fusarium species. For comparative purposes Fusarium roseum was included in this study as a well known producer of cyclic and linear fusigen.     

The Siderophore Metabolome of Azotobacter vinelandii

In this study, we performed a detailed characterization of the siderophore metabolome, or “chelome,” of the agriculturally important and widely studied model organism Azotobacter vinelandii. Using a new high-resolution liquid chromatography-mass spectrometry (LC-MS) approach, we found over 35 metal-binding secondary metabolites, indicative of a vast chelome in A. vinelandii. These include vibrioferrin, a siderophore previously observed only in marine bacteria. Quantitative analyses of siderophore production during diazotrophic growth with different sources and availabilities of Fe showed that, under all tested conditions, vibrioferrin was present at the highest concentration of all siderophores and suggested new roles for vibrioferrin in the soil environment. Bioinformatic searches confirmed the capacity for vibrioferrin production in Azotobacter spp. and other bacteria spanning multiple phyla, habitats, and lifestyles. Moreover, our studies revealed a large number of previously unreported derivatives of all known A. vinelandii siderophores and rationalized their origins based on genomic analyses, with implications for siderophore diversity and evolution. Together, these insights provide clues as to why A. vinelandii harbors multiple siderophore biosynthesis gene clusters. Coupled with the growing evidence for alternative functions of siderophores, the vast chelome in A. vinelandii may be explained by multiple, disparate evolutionary pressures that act on siderophore production.     

A comparison of Fe bioavailability and binding of a catecholate siderophore with virus-mediated lysates from the marine bacterium Vibrio alginolyticus PWH3a

We have examined the bioavailability of Fe complexed to a siderophore produced by the heterotrophic marine bacterium Vibrio alginolyticus isolate PWH3a and Fe from ligand-complexes present in virus-mediated lysates (using phage PWH3a-P1) of this same bacterium. Fe-binding functional groups, stability constants and the bioavailability of Fe associated with these two separate ligand pools were determined and contrasted to previous work. Under low-Fe growth conditions, axenic cultures of V. alginolyticus PWH3a were shown to produce catecholate siderophores, while neither catecholate nor hydroxamate-type Fe-binding moieties were detected in virus-generated cell lysates. Analysis of the overall binding strength using electrochemical techniques revealed that the siderophore-containing organic extract and the organics in the virus-mediated lysates had Fe-binding constants comparable to the weaker L₂-type ligands found throughout the water column in seawater. A further purification of the siderophore-containing extract revealed a ligand with a stability constant of logK′_FeL,Fe3+ = 22.3, typical for siderophores and L₁-type of ligands found in marine surface waters. In assimilation studies, the Fe in the lysate was found to be more bioavailable to our model heterotrophic bacterium, autotrophic cyanobacterium and eukaryotic diatom cultures than the catecholate siderophore produced by the Vibrio sp. This work demonstrates that the Fe-containing components of virus-mediated cell lysates are different than siderophores secreted by these same cells, and as such continues to build the argument supporting the importance of virus-mediated Fe regeneration in marine surface waters.