Role of antibiotics and siderophores in biocontrol of take-all disease of wheat

Both antibiotics and siderophores have been implicated in the control of soilborne plant pathogens by fluorescent pseudomonads. In Pseudomonas fluorescens 2–79, which suppresses take-all of wheat, the importance of the antibiotic phenazine-1-carboxylic acid was established with mutants deficient or complemented for antiobiotic production and by isolation of the antibiotic from the roots of wheat colonized by the bacteria. Genetic and biochemical studies of phenazine synthesis have focused on two loci; the first is involved in production of both anthranilic acid and phenazine-1-carboxylic acid, and the second encodes genes involved directly in phenazine synthesis. Because the antibiotic does not account fully for the suppressiveness of strain 2-79, additional mutants were analyzed to evaluate the role of the fluorescent siderophore and of an antifungal factor (Aff, identified as anthranilic acid) that accumulates when iron is limiting. Whereas strains producing only the siderophore conferred little protection against take-all, Aff⁺ strains were suppressive, but much less so than phenazine-producing strains. Iron-regulated nonsiderophore antibiotics may be produced by fluorescent pseudomonads more frequently than previously recognized, and could be partly responsible for beneficial effects that were attributed in the past to fluorescent siderophores.     

Elucidation of the complete ferrichrome A biosynthetic pathway in Ustilago maydis

Iron is an important element for many essential processes in living organisms. To acquire iron, the basidiomycete Ustilago maydis synthesizes the iron‐chelating siderophores ferrichrome and ferrichrome A. The chemical structures of these siderophores have been elucidated long time ago but so far only two enzymes involved in their biosynthesis have been described. Sid1, an ornithine monoxygenase, is needed for the biosynthesis of both siderophores, and Sid2, a non‐ribosomal peptide synthetase (NRPS), is involved in ferrichrome generation. In this work we identified four novel enzymes, Fer3, Fer4, Fer5 and Hcs1, involved in ferrichrome A biosynthesis in U. maydis. By HPLC‐MS analysis of siderophore accumulation in culture supernatants of deletion strains, we show that Fer3, an NRPS, Fer4, an enoyl‐coenzyme A (CoA)‐hydratase, and Fer5, an acylase, are required for ferrichrome A production. We demonstrate by conditional expression of the hydroxymethyl glutaryl (HMG)‐CoA synthase Hcs1 in U. maydis that HMG‐CoA is an essential precursor for ferrichrome A. In addition, we heterologously expressed and purified Hcs1, Fer4 and Fer5, and demonstrated the enzymatic activities by in vitro experiments. Thus, we describe the first complete fungal siderophore biosynthetic pathway by functionally characterizing four novel genes responsible for ferrichrome A biosynthesis in U. maydis.     

棘孢木霉嗜铁素sidA基因敲除及表型分析

明确棘孢木霉菌Trichoderma asperellum嗜铁素合成的关键基因,能够为进一步探索嗜铁素在生防和促生中的作用奠定基础。本研究在对sidA基因进行定位、结构分析和RT-PCR检测的基础上,利用double-joint PCR技术构建基因敲除载体,经聚乙二醇（PEG）介导原生质体转化、潮霉素初筛、PCR和southern blot验证获得突变株,并对其表型进行分析,获得2株性能稳定的敲除突变体?sidA1和?sidA2。与野生型相比,?sidA1和?sidA2的嗜铁素产量在5d时分别下降了38.67%和36.65%;孢子萌发率在12h时分别下降了45.33%和47.47%;产孢量在10d时分别下降了33.01%和41.02%,且突变株在受NaCl、KCl、SDS等胁迫时的抗性较野生型降低。表明sidA基因的缺失降低了嗜铁素产量,抑制了菌体的生长以及对胁迫因子的抗性,sidA基因是影响棘孢木霉嗜铁素产生的关键基因之一。     

Interactions between iron availability, aluminium toxicity and fungal siderophores

The influence of iron, aluminium and of the combined application of both metals on microbial biomass and production of siderophores by three fungi (Aspergillus nidulans, Neurospora crassa and Hymenoscyphus ericae) were investigated. All three species showed a strong iron regulation and Al-sensitivity of siderophore biosynthesis although several differences remained species dependent. Inhibitory effects of Fe and Al on siderophore-production were additive and the higher binding capacity of siderophores towards iron could be compensated by a higher Al-availability. Although pH itself is also important for regulation of siderophore biosynthesis, an indirect effect of Al on siderophore production via an Al-induced pH decrease could be outlined. The toxic effects of Al resulting in a reduced biomass production were compensated by high Fe-availability, whereas the addition of DFAM, a bacterial siderophore, enhanced Al-toxicity.     

Siderophore-mediated strategies of iron acquisition by extraintestinal isolates of Enterobacter spp

A total of 89 examined Enterobacter isolates belonging to three species: E. cloaceae, E. aerogenes and E. sakazakii, produced iron chelators detected in universal CAS assay. In chemical assays the strains were shown to excrete mostly catecholate (88 strains) and hydroxamate (42 strains) type of siderophores. Forty-one strains produced both catecholate and hydroxamate siderophores whereas one isolate produced only hydroxamate. Besides, the isolates were screened for genes coding for another siderophore: yersiniabactin. The genes for biosynthesis and uptake of yersiniabactin are located on the high-pathogenicity island (HPI) of Yersinia spp. The presence of three marker genes irp1, irp2 and fyuA was estimated by polymerase chain reaction. Two strains: E. aerogenes and E. cloaceae possessed irp1, irp2 and fyuA genes. PCR products of irp1, irp2 and fyuA were of 240, 280 and 780 bp, respectively.     

DNA homology between siderophore genes from fluorescent pseudomonads.

Many species of pseudomonads produce fluorescent siderophores involved in iron uptake. We have investigated the DNA homology between the siderophore synthesis genes of an opportunist animal pathogen, Pseudomonas aeruginosa, and three plant-associated species Pseudomonas syringae, Pseudomonas putida and Pseudomonas sp. B10. There is extensive homology between the DNA from the different species, consistent with the suggestion that the different siderophore synthesis genes have evolved from the same ancestral set of genes. The existence of DNA homology allowed us to clone some of the siderophore synthesis genes from P. aeruginosa, and genetic mapping indicates that the cloned DNA lies in a locus previously identified as being involved in siderophore production.     

Characterization of staphyloferrin A biosynthetic and transport mutants in Staphylococcus aureus

Iron is critical for virtually all forms of life. The production of high-affinity iron chelators, siderophores, and the subsequent uptake of iron–siderophore complexes are a common strategy employed by microorganisms to acquire iron. Staphylococcus aureus produces siderophores but genetic information underlying their synthesis and transport is limited. Previous work implicated the sbn operon in siderophore synthesis and the sirABC operon in uptake. Here we characterize a second siderophore biosynthetic locus in S. aureus ; the locus consists of four genes (in strain Newman these open reading frames are designated NWMN_2079–2082) which, together, are responsible for the synthesis and export of staphyloferrin A, a polycarboxylate siderophore. While deletion of the NWMN_2079–2082 locus did not affect iron-restricted growth of S. aureus , strains bearing combined sbn and NWMN_2079–2082 locus deletions produced no detectable siderophore and demonstrated severely attenuated iron-restricted growth. Adjacent to NWMN_2079–2082 resides the htsABC operon, encoding an ABC transporter previously implicated in haem acquisition. We provide evidence here that HtsABC, along with the FhuC ATPase, is required for the uptake of staphyloferrin A. The crystal structure of apo-HtsA was determined and identified a large positively charged region in the substrate-binding pocket, in agreement with a role in binding of anionic staphyloferrin A.     

Proteobactin and a yersiniabactin-related siderophore mediate iron acquisition in Proteus mirabilis

Proteus mirabilis causes complicated urinary tract infections (UTIs). While the urinary tract is an iron-limiting environment, iron acquisition remains poorly characterized for this uropathogen. Microarray analysis of P. mirabilis HI4320 cultured under iron limitation identified 45 significantly upregulated genes (P ≤ 0.05) that represent 21 putative iron-regulated systems. Two gene clusters, PMI0229-0239 and PMI2596-2605, encode putative siderophore systems. PMI0229-0239 encodes a non-ribosomal peptide synthetase-independent siderophore system for producing a novel siderophore, proteobactin. PMI2596-2605 are contained within the high-pathogenicity island, originally described in Yersinia pestis, and encodes proteins with apparent homology and organization to those involved in yersiniabactin production and uptake. Cross-feeding and biochemical analysis shows that P. mirabilis is unable to utilize or produce yersiniabactin, suggesting that this yersiniabactin-related locus is functionally distinct. Only disruption of both systems resulted in an in vitro iron-chelating defect; demonstrating production and iron-chelating activity for both siderophores. These findings clearly show that proteobactin and the yersiniabactin-related siderophore function as iron acquisition systems. Despite the activity of both siderophores, only mutants lacking the yersiniabactin-related siderophore have reduced fitness in vivo. The fitness requirement for the yersiniabactin-related siderophore during UTI shows, for the first time, the importance of siderophore production in vivo for P. mirabilis.     

Gene cluster for ferric iron uptake in Agrobacterium tumefaciens MAFF301001

Agrobacterium tumefaciens harboring a Ti plasmid causes crown gall disease in dicot plants by transferring its T-DNA into plant chromosomes. Iron acquisition plays an important role for pathogenicity in animal pathogens and several phytopathogens and for growth in the rhizosphere and on plant surfaces. Under iron-limiting condition, bacteria produce various iron-chelating agents called siderophores. Agrobacterium strains have the diversity in producing siderophores and a certain strain produces a typical catechol-type siderophore, called agrobactin, although its biosynthesis genes have not been analyzed yet. Here we describe the cloning and characterization of a functional gene cluster involved in ferric iron uptake in A. tumefaciens strain MAFF301001. Four complete open reading frames (ORFs) were found in 5-kb region of a genomic library clone 1A3. We named these genes agb, after agrobactin. agbC, agbE, agbB and agbA genes were identified in this order, and narrow intergenic spaces suggested that these genes constitute an operon. Predicted agb gene products and their phylogenetic analysis showed sequence similarity with enzymes which are involved in ferric iron uptake in other bacteria. Southern hybridization analysis clearly indicated the location of agb genes on the linear chromosome in strain MAFF301001 but the complete lack in another A. tumefaciens strain C58. Mutation analysis of agbB revealed that it is essential for growth and production of catechol compounds in iron-limiting medium.     

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

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

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.     

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.