Disruption of two genes for chitin synthase in the phytopathogenic fungus Ustilago maydis

The phytopathogenic fungus Ustilago maydis exhibits a dimorphic transition in which non-pathogenic, yeast-like cells mate to form a pathogenic, filamentous dikaryon. Northern analysis indicated that two chitin synthase genes, chs1 and chs2, from U. maydis are expressed at similar levels in yeast-like cells and in cells undergoing the mating reaction leading to the filamentous cell type. A mutation was constructed in each of the chitin synthase genes by targeted gene disruption. Each mutant showed a reduction in the level of trypsin-activated enzyme activity, compared with a wild-type strain, but retained the wild-type morphology, the ability to mate and the ability to form the filamentous pathogenic cell type.     

Characterization of two novel non-reducing polyketide synthase genes from the lichen-forming fungus Hypogymnia physodes

Lichens are known to produce a variety of secondary metabolites including polyketides, which have valuable biological activities. Some polyketides are produced solely by lichens. The biosynthesis of these compounds is primarily governed by iterative type I polyketide synthases. Hypogymnia physodes synthesize polyketides such as physodic, physodalic and hydroxyphysodic acid and atranorin, which are non-reducing polyketides. Two novel non-reducing polyketide synthase (PKS) genes were isolated from a fosmid genomic library of a mycobiont of H. physodes using a 409bp fragment corresponding to part of the reductase (R) domain as a probe. H. physodes PKS1 (Hyopks1) and PKS2 (Hypopks2) contain keto synthase (KS), acyl transferase (AT), acyl carrier protein (ACP), methyl transferase (ME) and R domains. Classification based on phylogeny analysis using the translated KS and AT domains demonstrated that Hypopks1 and Hypopks2 are members of the fungal non-reducing PKSs clade III. This is the first report of non-reducing PKSs containing the R domain-mediated release mechanisms in lichens, which are also rare fungal type I PKS in non-lichenized filamentous fungi.     

Diversity of type I polyketide synthase genes in the wood-decay fungus Xylaria sp. BCC 1067

Fungal type I polyketide (PK) compounds are highly valuable for medical treatment and extremely diverse in structure, partly because of the enzymatic activities of reducing domains in polyketide synthases (PKSs). We have cloned several PKS genes from the fungus Xylaria sp. BCC 1067, which produces two polyketides: depudecin (reduced PK) and 19,20-epoxycytochalasin Q (PK-nonribosomal peptide (NRP) hybrid). Two new degenerate primer sets, KA-series and XKS, were designed to amplify reducing PKS and PKS-NRP synthetase hybrid genes, respectively. Five putative PKS genes were amplified in Xylaria using KA-series primers and two more with the XKS primers. All seven are predicted to encode proteins homologous to highly reduced (HR)-type PKSs. Previously designed primers in LC-, KS-, and MT-series identified four additional PKS gene fragments. Selected PKS fragments were used as probes to identify PKS genes from the genomic library of this fungus. Full-length sequences for five PKS genes were obtained: pks12, pks3, pksKA1, pksMT, and pksX1. They are structurally diverse with 1-9 putative introns and products ranging from 2162 to 3654 amino acids in length. The finding of 11 distinct PKS genes solely by means of PCR cloning supports that PKS genes are highly diverse in fungi. It also indicates that our KA-series primers can serve as powerful tools to reveal the genetic potential of fungi in production of multiple types of HR PKs, which the conventional compound screening could underestimate.     

Characterization of two polyketide synthase genes in Exophiala lecanii-corni, a melanized fungus with bioremediation potential

Exophiala lecanii-corni has significant bioremediation potential because it can degrade a wide range of volatile organic compounds. In order to identify sites for the insertion of genes that might enhance this potential, a genetic analysis of E. lecanii-corni was undertaken. Two polyketide synthase genes, ElPKS1 and ElPKS2, have now been discovered by a PCR-based strategy. ElPKS1 was isolated by a marker rescue technique. The nucleotide sequence of ElPKS1 consists of a 6576-bp open reading frame encoding a protein with 2192 amino acids, which was interrupted by a 60-bp intron near the 5' end and a 54-bp intron near the 3' end. Sequence analysis, results from disruption experiments, and physiological tests showed that ElPKS1 encoded a polyketide synthase required for melanin biosynthesis. Since ElPKS1 is non-essential, it is a desirable bioengineering target site for the insertion of native and foreign genes. The successful expression of these genes could enhance the bioremediation capability of the organism. ElPKS2 was cloned by colony hybridization screening of a partial genomic library with an ElPKS2 PCR product. ElPKS2 had a 6465-bp open reading frame that encoded 2155 amino acids and had introns of 56, 67, 54, and 71 bp. Although sequence analysis of the derived protein of ElPKS2 confirmed the polyketide synthase nature of its protein product, the function of that product remains unclear.     

Functional analysis of the polyketide synthase genes in the filamentous fungus Gibberella zeae (anamorph Fusarium graminearum)

Polyketides are a class of secondary metabolites that exhibit a vast diversity of form and function. In fungi, these compounds are produced by large, multidomain enzymes classified as type I polyketide synthases (PKSs). In this study we identified and functionally disrupted 15 PKS genes from the genome of the filamentous fungus Gibberella zeae. Five of these genes are responsible for producing the mycotoxins zearalenone, aurofusarin, and fusarin C and the black perithecial pigment. A comprehensive expression analysis of the 15 genes revealed diverse expression patterns during grain colonization, plant colonization, sexual development, and mycelial growth. Expression of one of the PKS genes was not detected under any of 18 conditions tested. This is the first study to genetically characterize a complete set of PKS genes from a single organism.     

Localization of polyketide synthase encoding genes to the toxic dinoflagellate Karenia brevis

Karenia brevis is a toxic marine dinoflagellate endemic to the Gulf of Mexico. Blooms of this harmful alga cause fish kills, marine mammal mortalities and neurotoxic shellfish poisonings. These harmful effects are attributed to a suite of polyketide secondary metabolites known as the brevetoxins. The carbon framework of all polyketides is assembled by a polyketide synthase (PKS). Previously, PKS encoding genes were amplified from K. brevis culture and their similarity to a PKS gene from the closely related protist, Cryptosporidium parvum, suggested that these genes originate from the dinoflagellate. However, K. brevis has not been grown axenically. The associated bacteria might be the source of the toxins or the PKS genes. Herein we report the localization of PKS encoding genes by a combination of flow cytometry/PCR and fluorescence in situ hybridization (FISH). Two genes localized exclusively to K. brevis cells while a third localized to both K. brevis and associated bacteria. While these genes have not yet been linked to toxin production, the work describes the first definitive evidence of resident PKS genes in any dinoflagellate.     

Characterization of a mutant strain of a filamentous fungus Cladosporium phlei for the mass production of the secondary metabolite phleichrome

UV-mutagenesis was performed to obtain mutant strains that demonstrate altered production of phleichrome, a secondary metabolite of Cladosporium phlei. Among fifty mutants selected, based on the increased area and intensity of the purple pigment surrounding the colonies, the strain M0035 showed the highest production of phleichrome, more than seven fold over wild type. Plate cultures of the M0035 strain resulted in a total of 592 mg phleichrome consisting of 146 mg and 446 mg from the mycelia and agar media, respectively. The M0035 strain displayed a growth rate and a mycelial mass comparable to the parental strain but had significantly reduced asexual sporulation.     

Natural biocombinatorics in the polyketide synthase genes of the actinobacterium Streptomyces avermitilis

Modular polyketide synthases (PKSs) of bacteria provide an enormous reservoir of natural chemical diversity. Studying natural biocombinatorics may aid in the development of concepts for experimental design of genes for the biosynthesis of new bioactive compounds. Here we address the question of how the modularity of biosynthetic enzymes and the prevalence of multiple gene clusters in Streptomyces drive the evolution of metabolic diversity. The phylogeny of ketosynthase (KS) domains of Streptomyces PKSs revealed that the majority of modules involved in the biosynthesis of a single compound evolved by duplication of a single ancestor module. Using Streptomyces avermitilis as a model organism, we have reconstructed the evolutionary relationships of different domain types. This analysis suggests that 65% of the modules were altered by recombinational replacements that occurred within and between biosynthetic gene clusters. The natural reprogramming of the biosynthetic pathways was unambiguously confined to domains that account for the structural diversity of the polyketide products and never observed for the KS domains. We provide examples for natural acyltransferase (AT), ketoreductase (KR), and dehydratase (DH)–KR domain replacements. Potential sites of homologous recombination could be identified in interdomain regions and within domains. Our results indicate that homologous recombination facilitated by the modularity of PKS architecture is the most important mechanism underlying polyketide diversity in bacteria.     

Isolation of ten microsatellite loci in an EST library of the phytopathogenic fungus Puccinia striiformis f.sp. tritici

We report the characterization of ten microsatellite markers in the fungus Puccinia striiformis f.sp. tritici, responsible for yellow rust disease on wheat. A published EST library was scanned for microsatellite motives, and over 15 selected EST sequences, 13 were successfully amplified and ten exhibited polymorphism over an international collection of 43 isolates. These new microsatellites, added to the few previously published ones, provide a sufficient set of markers to perform population genetic studies.     

Isolation and characterization of a non-reducing polyketide synthase gene from the lichen-forming fungus Usnea longissima

Usnea longissima has long been used as a traditional medicine in China, India, Turkey, Canada and Europe. This lichen can produce several bioactive compounds that primarily belong to the polyketide family. The enzymes responsible for the production of these compounds are the polyketide synthases, but the biosynthetic processes in lichens are still unclear. In this study, a cultured mycobiont of Usnea longissima was used to isolate and characterize a polyketide synthase gene (UlPKS1). Complete sequence information regarding UlPKS1 (6,468 bp) was obtained by screening a Fosmid genomic library using a 512-bp fragment corresponding to part of the ketosynthase (KS) domain. Sequence analysis of UlPKS1 suggested that it contained features of a non-reducing fungal type I PKS with a starter unit of ACP transacylase (SAT), ketosynthase (KS), product template (PT), acyl carrier protein (ACP) transacylase, acyltransferase (AT) and thioesterase (TE) domain, and had five intervening introns. The domain organization of UlPKS1 (SAT-KS-AT-PT-ACP-ACP-TE) was quite similar to that of aromatic PKSs, and phylogenetic analysis showed that UlPKS1 belonged to the clade of lichenized fungal non-reducing PKS. RT-PCR analyses revealed that the expression of UlPKS1 was down-regulated by glycine and high concentrations of sorbitol, inositol and fructose and up-regulated by sucrose and glucose. Here, we introduce a non-reducing PKS gene in the lichen-forming fungus U. longissima, with a domain structure similar to the structure of orsellinic acid synthase A (OrsA) which is required for orsellinic acid biosynthesis in Aspergillus nidulans.     

Isolation and characterization of a reducing polyketide synthase gene from the lichen-forming fungus Usnea longissima

The reducing polyketide synthases found in filamentous fungi are involved in the biosynthesis of many drugs and toxins. Lichens produce bioactive polyketides, but the roles of reducing polyketide synthases in lichens remain to be clearly elucidated. In this study, a reducing polyketide synthase gene (U1PKS3) was isolated and characterized from a cultured mycobiont of Usnea longissima. Complete sequence information regarding U1PKS3 (6,519 bp) was obtained by screening a fosmid genomic library. A U1PKS3 sequence analysis suggested that it contains features of a reducing fungal type I polyketide synthase with β-ketoacyl synthase (KS), acyltransferase (AT), dehydratase (DH), enoyl reductase (ER), ketoacyl reducatse (KR), and acyl carrier protein (ACP) domains. This domain structure was similar to the structure of ccRadsl, which is known to be involved in resorcylic acid lactone biosynthesis in Chaetomium chiversii. The results of phylogenetic analysis located U1PKS3 in the clade of reducing polyketide synthases. RT-PCR analysis results demonstrated that UIPKS3 had six intervening introns and that UIPKS3 expression was upregulated by glucose, sorbitol, inositol, and mannitol.     

Complex organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase.

The avermectin (Av) polyketide synthase (PKS) and erythromycin (Er) PKS are encoded by modular repeats of DNA, but the genetic organization of the modules encoding Av PKS is more complex than Er PKS. Sequencing of several related DNA fragments from Streptomyces avermitilis that are part of the Av biosynthetic gene cluster, revealed that they encode parts of large multifunctional PKS proteins. The Av PKS proteins show strong similarity to each other, as well as similarity to Er PKS proteins [Donadio et al., Science 252 (1991) 675-679] and fatty acid synthases. Partial DNA sequencing of the 65-kb region containing all the related sequence elements in the avr genes provides evidence for twelve modular repeats encoding FAS-like domains. The genes encoding the Av PKS are organized as two sets of six modular repeats which are convergently transcribed.     

Adaptation of the phytopathogenic fungus Fusarium decemcellulare to oxidative stress

The adaptive response of the phytopathogenic fungus Fusarium decemcellulare to the oxidative stress induced by hydrogen peroxide and juglone (5-hydroxy-1,4-naphthoquinone) was studied. At concentrations higher than 1 mM, H2O2 and juglone completely inhibited the growth of the fungus. The 60-min pretreatment of logarithmic-phase cells with nonlethal concentrations of H2O2 (0.25 mM) and juglone (0.1 mM) led to the development of a resistance to high concentrations of these oxidants. The stationary-phase cells were found to be more resistant to the oxidants than the logarithmic-phase cells. The adaptation of fungal cells to H2O2 and juglone was associated with an increase in the activity of cellular catalase and superoxide dismutase, the main oxidative stress defense of enzymes.     

Isolation of metabolic genes and demonstration of gene disruption in the phytopathogenic fungus Ustilago maydis

A cDNA library was constructed in the yeast expression vector pYcDE8 using mRNA from the phytopathogenic fungus Ustilago maydis and cDNAs capable of complementing mutations in three yeast genes, URA3, LEU2 and TPI1, were identified. Nucleotide sequence analysis indicated that the cDNA clone, which complemented the yeast ura3 mutation, carries the pyr6 gene encoding orotidine-5'-phosphate decarboxylase. The genomic copy of the pyr6 gene was isolated by hybridization with the cDNA and used to complement a pyr- mutant of U. maydis. One-step gene disruption was demonstrated by transforming U. maydis with a copy of the pyr6 gene interrupted in the coding region by a selectable marker for resistance to hygromycin B.     

Identification and characterization of the niddamycin polyketide synthase genes from Streptomyces caelestis.

The genes encoding the polyketide synthase (PKS) portion of the niddamycin biosynthetic pathway were isolated from a library of Streptomyces caelestis NRRL-2821 chromosomal DNA. Analysis of 40 kb of DNA revealed the presence of five large open reading frames (ORFs) encoding the seven modular sets of enzymatic activities required for the synthesis of a 16-membered lactone ring. The enzymatic motifs identified within each module were consistent with those predicted from the structure of niddamycin. Disruption of the second ORF of the PKS coding region eliminated niddamycin production, demonstrating that the cloned genes are involved in the biosynthesis of this compound.     

Rapid screening of a large insert BAC library for specific 16S rRNA genes using TRFLP

It is widely believed that the vast majority of microbes in the environment have-yet-to-be cultured using standard techniques. Bulk DNA from microbial communities is therefore often cloned into large insert vectors (e.g. bacterial artificial chromosomes [BAC] or cosmids) in order to study the genetic properties of these as yet (un)-cultured bacteria. In a typical BAC experiment, tens of thousands of clones are generated with only a small fraction of colonies containing the target(s) of interest. Efficient screening methodologies are therefore needed to allow targeted clone isolation. In this paper, we describe a rapid, inexpensive protocol that allows for the identification of specific 16S ribosomal RNA genes in a metagenomic library arrayed into 384-well microtiter plates. The rapid screening protocol employs Terminal Restriction Fragment Length Polymorphism (TRFLP) analysis to identify wells containing specific T-RF peaks. A nested approach using multiplexed samples of 384, 48, 8, and single colony analysis is described and applied in order to survey a BAC library generated from a marine microbial community off the coast of New Jersey. Screening revealed a total of 50 different 16 rRNA genes within the BAC library. Overall, the multiplexing format provided a simple, cost effective methodology for detecting clones bearing a target gene of interest in a large clone library. However, the limitations of screening BAC libraries using PCR methodologies and recommendations for improved screening efficiency using this approach are also discussed.     

Isolation and disruption of the melanin pathway polyketide synthase gene of the softwood deep stain fungus Ceratocystis resinifera

Ceratocystis resinifera hyphae produce a black melanin pigment causing a deep stain in softwood logs. We exploited the homology of polyketide synthases to clone PKS1, a gene responsible for dihydroxynaphthalene-melanin biosynthesis in C. resinifera. Sequence analysis indicated that PKS1 has two introns near its 5(') end and encodes a 2188-amino acid polypeptide with five functional domains: beta-ketoacyl synthase, acyl transferase, two acyl carrier proteins and a thioesterase/Claisen cyclase. A gene disruption construct designed to replace a portion of PKS1 with a hygromycin resistance cassette was transformed into C. resinifera through Agrobacterium tumefaciens-mediated transformation. PKS1 null mutants had an albino phenotype, and pigmentation was restored by the addition of scytalone, a melanin pathway intermediate. The disruption of PKS1 and restoration of pigmentation with scytalone confirmed the presence of a dihydroxynaphthalene-melanin pathway in C. resinifera. The transformation method described in this paper is the first reported for a Ceratocystis species.