Chromosomal organization of TOX2, a complex locus controlling host-selective toxin biosynthesis in Cochliobolus carbonum.

Race 1 isolates of the filamentous fungus Cochliobolus carbonum are exceptionally virulent on certain genotypes of maize due to production of a cyclic tetrapeptide, HC-toxin. In crosses between toxin-producing (Tox2+) and toxin-nonproducing (Tox2-) isolates, toxin production segregates in a simple 1:1 pattern, suggesting the involvement of a single genetic locus, which has been named TOX2. Earlier work had shown that in isolate SB111, TOX2 consists in part of two copies of a gene, HTS1, that encodes a 570-kD cyclic peptide synthetase and is lacking in Tox2- isolates. The genomic structure of TOX2 and the relationship between the two copies of HTS1 have now been clarified by using pulsedfield gel electrophoresis and physical mapping. In isolate SB111, both copies of HTS1 are on the largest chromosome (3.5 Mb), which is not present in the related Tox2- strain SB114. Two other genes known or thought to be important for HC-toxin biosynthesis, TOXA and TOXC, are also on the same chromosome in multiple copies. Other independent Tox2+ isolates also have two linked copies of HTS1, but in some isolates the size of the chromosome containing HTS1 is 2.2 Mb. Evidence obtained with Tox2+ -unique and with random probes is consistent with a reciprocal translocation as the cause of the difference in the size of the HTS1-containing chromosome among the Tox2+ isolates studied here. Physical mapping of the 3.5-Mb chromosome of SB111 that contains HTS1 using rare-cutting restriction enzymes and engineered restriction sites was used to map the chromosome location of the two copies of HTS1 and the three copies of TOXC. The results indicate that TOX2 is a complex locus that extends over more than 500 kb. The capacity to produce HC-toxin did not evolve by any single, simple mechanism.     

Reduced virulence caused by meiotic instability of the TOX2 chromosome of the maize pathogen Cochliobolus carbonum

The mechanisms by which pathogenic fungi evolve are poorly understood. Production of the host-selective cyclic peptide HC-toxin is controlled by a complex locus, TOX2, in the plant pathogen Cochliobolus carbonum. Crosses between toxin-producing (Tox2+) and toxin-nonproducing (Tox2-) isolates, as well as crosses between isolates in which the TOX2 genes were on chromosomes of different size, yielded progeny that had lost one or more copies of one or more of the TOX2 genes. Of approximately 200 progeny analyzed, eight (4%) had lost at least one TOX2 gene. All of them still had at least one functional copy of all of the known genes required for HC-toxin production (HTS1, TOXA, TOXC, and TOXE). Most deletion strains could be explained by simple chromosome breaks resulting in the loss of major contiguous portions (0.8 to 1.4 Mb) of the 3.5-Mb TOX2 chromosome, whereas others had more complicated patterns. All deletion strains had normal growth and were fertile, indicating that the 1.4 Mb of DNA contained no essential housekeeping genes. Most strains were also still virulent (Tox2+), but two had a novel phenotype of reduced virulence (RV), characterized by smaller lesions that expanded at a reduced rate and an inability to colonize plants systemically. Although the RV strains made no detectable HC-toxin in culture, the RV phenotype was dependent on the presence of a functional copy of HTS1, which encodes the central enzyme in HC-toxin biosynthesis. We propose that the RV strains still make a low level of HC-toxin, at least in planta, and that this is due to the loss of one or more genes that contribute to, but are not absolutely required for, HC-toxin synthesis.     

Regulation of cyclic peptide biosynthesis and pathogenicity in Cochliobolus carbonum by TOXEp, a novel protein with a bZIP basic DNA-binding motif and four ankyrin repeats

HC-toxin is an epoxide-containing cyclic tetrapeptide that is a critical virulence determinant in the pathogenic interaction between the filamentous fungus Cochliobolus carbonum and maize. HC-toxin exerts a potent cytostatic effect on plant and animal cells by inhibiting histone deacetylase. The biosynthesis of HC-toxin by C. carbonum is controlled by a complex genetic locus, TOX2, that contains multiple, duplicated copies of genes encoding export and biosynthetic enzymes. A new gene in the TOX2 complex, TOXE, has now been isolated. Mutation of TOXE by targeted gene disruption has no effect on growth and sporulation but abolishes HC-toxin production and pathogenicity. TOXE is required for the expression of three genes with a known or putative role in HC-toxin production, but is not required for expression of HTS1, which encodes the large, multifunctional peptide synthetase that is the central enzyme in HC-toxin biosynthesis. At its N-terminus, TOXEp has a bZIP basic DNA binding domain, but it does not contain any discernible leucine zipper or helix-loop-helix. At its carboxy terminus, TOXEp contains four ankyrin repeats. In having these two common regulatory motifs in a single polypeptide, TOXEp appears to represent a novel class of regulatory protein. TOXE is present only in HC-toxin-producing (Tox2⁺) isolates of C. carbonum. Most Tox2⁺ isolates have two copies; in strain SB111, one copy of TOXE is on the same 3.5-Mb chromosome that contains all of the other genes known to be involved in HC-toxin biosynthesis, and the second copy of TOXE is on a 0.7-Mb chromosome. Received: 20 April 1998 / Accepted: 21 September 1998     

Regulation of cyclic peptide biosynthesis and pathogenicity in Cochliobolus carbonum by TOXEp, a novel protein with a bZIP basic DNA-binding motif and four ankyrin repeats

HC-toxin is an epoxide-containing cyclic tetrapeptide that is a critical virulence determinant in the pathogenic interaction between the filamentous fungus Cochliobolus carbonum and maize. HC-toxin exerts a potent cytostatic effect on plant and animal cells by inhibiting histone deacetylase. The biosynthesis of HC-toxin by C. carbonum is controlled by a complex genetic locus, TOX2, that contains multiple, duplicated copies of genes encoding export and biosynthetic enzymes. A new gene in the TOX2 complex, TOXE, has now been isolated. Mutation of TOXE by targeted gene disruption has no effect on growth and sporulation but abolishes HC-toxin production and pathogenicity. TOXE is required for the expression of three genes with a known or putative role in HC-toxin production, but is not required for expression of HTS1, which encodes the large, multifunctional peptide synthetase that is the central enzyme in HC-toxin biosynthesis. At its N-terminus, TOXEp has a bZIP basic DNA binding domain, but it does not contain any discernible leucine zipper or helix-loop-helix. At its carboxy terminus, TOXEp contains four ankyrin repeats. In having these two common regulatory motifs in a single polypeptide, TOXEp appears to represent a novel class of regulatory protein. TOXE is present only in HC-toxin-producing (Tox2⁺) isolates of C. carbonum. Most Tox2⁺ isolates have two copies; in strain SB111, one copy of TOXE is on the same 3.5-Mb chromosome that contains all of the other genes known to be involved in HC-toxin biosynthesis, and the second copy of TOXE is on a 0.7-Mb chromosome.     

Mutational analysis of beta-glucanase genes from the plant-pathogenic fungus Cochliobolus carbonum

Two new beta-glucanase-encoding genes, EXG2 and MLG2, were isolated from the plant-pathogenic fungus Cochliobolus carbonum using polymerase chain reaction based on amino acid sequences from the purified proteins. EXG2 encodes a 46.6-kDa exo-beta1,3-glucanase and is located on the same 3.5-Mb chromosome that contains the genes of HC-toxin biosynthesis. MLG2 encodes a 26.8-kDa mixed-linked (beta1,3-beta1,4) glucanase with low activity against beta1,4-glucan and no activity against beta1,3-glucan. Specific mutants of EXG2 and MLG2 were constructed by targeted gene replacement. Strains with multiple mutations (genotypes exg1/mlg1, exg2/mlg1, mlg1/mlg2, and exg1/exg2/mlg1/mlg2) were also constructed by sequential disruption and by crossing. Total mixed-linked glucanase activity in culture filtrates of mlg1/mlg2 and exg1/exg2/mlg1/mlg2 mutants was reduced by approximately 73%. Total beta1,3-glucanase activity was reduced by 10, 54, and 96% in exg2, mlg1, and exg1/exg2/mlg1/mlg2 mutants, respectively. The quadruple mutant showed only a modest decrease in growth on beta1,3-glucan or mixed-linked glucan. None of the mutants showed any decrease in virulence.     

A eukaryotic alanine racemase gene involved in cyclic peptide biosynthesis

The cyclic tetrapeptide HC-toxin is an essential virulence determinant for the plant pathogenic fungus Cochliobolus carbonum and an inhibitor of histone deacetylase. The major form of HC-toxin contains the D-isomers of Ala and Pro. The non-ribosomal peptide synthetase that synthesizes HC-toxin has only one epimerizing domain for conversion of L-Pro to D-Pro; the source of D-Ala has remained unknown. Here we present the cloning and characterization of a new gene involved in HC-toxin biosynthesis, TOXG. TOXG is present only in HC-toxin-producing (Tox2(+)) isolates of C. carbonum. TOXG is able to support D-Ala-independent growth of a strain of Escherichia coli defective in D-Ala synthesis. A C. carbonum strain with both of its copies of TOXG mutated grows normally in culture, and although it no longer makes the three forms of HC-toxin that contain D-Ala, it still makes a minor form of HC-toxin that contains Gly in place of D-Ala. The addition of D-Ala to the culture medium restores production of the D-Ala-containing forms of HC-toxin by the toxG mutant. The toxG mutant has only partially reduced virulence. It is concluded that TOXG encodes an alanine racemase whose function is to synthesize D-Ala for incorporation into HC-toxin.     

The cyclic peptide synthetase catalyzing HC-toxin production in the filamentous fungus Cochliobolus carbonum is encoded by a 15.7-kilobase open reading frame.

Race 1 of Cochliobolus carbonum, a fungal plant pathogen, owes its exceptional virulence on certain genotypes of maize to the production of HC-toxin, a cyclic tetrapeptide. Production of HC-toxin is controlled by a single known gene, TOX2. Race 1, but not races that do not make HC-toxin, contains two copies of a 22-kilobase (kb) region of chromosomal DNA that is required for HC-toxin biosynthesis and hence virulence. We have sequenced this 22-kb region and here show that it contains an open reading frame of 15.7 kb that encodes a multifunctional cyclic peptide synthetase of potential M(r)574,620. This gene, called HTS1, apparently contains no introns. The predicted gene product, HC-toxin synthetase (HTS), contains four amino acid-binding (adenylate-forming) domains that are highly similar to those found in other cyclic peptide synthetases and other adenylate-binding enzymes. The DNA sequence encodes tryptic peptides derived from two HC-toxin biosynthetic enzymes, HC-toxin synthetase 1 (HTS-1) and HC-toxin synthetase 2 (HTS-2), indicating that these two enzymes exist in vivo as part of a single polypeptide. Consistent with this, in some enzyme preparations antibodies against the enzyme HTS-2, which was originally purified as a protein with a subunit M(r) of 160,000, recognize a protein with an estimated subunit M(r) greater than 480,000.     

A Biochemical Phenotype for a Disease Resistance Gene of Maize

In maize, major resistance to the pathogenic fungus Cochliobolus (Helminthosporium) carbonum race 1 is determined by the dominant allele of the nuclear locus hm. The interaction between C. carbonum race 1 and maize is mediated by a pathogen-produced, low molecular weight compound called HC-toxin. We recently described an enzyme from maize, called HC-toxin reductase, that inactivates HC-toxin by pyridine nucleotide-dependent reduction of an essential carbonyl group. We now report that this enzyme activity is detectable only in extracts of maize that are resistant to C. carbonum race 1 (genotype Hm/Hm or Hm/hm). In several genetic analyses, in vitro HC-toxin reductase activity was without exception associated with resistance to C. carbonum race 1. The results indicate that detoxification of HC-toxin is the biochemical basis of Hm-specific resistance of maize to infection by C. carbonum race 1.     

A Gene Related to Yeast HOS2 Histone Deacetylase Affects Extracellular Depolymerase Expression and Virulence in a Plant Pathogenic Fungus

A gene, HDC1, related to the Saccharomyces cerevisiae histone deacetylase (HDAC) gene HOS2, was isolated from the filamentous fungus Cochliobolus carbonum, a pathogen of maize that makes the HDAC inhibitor HC-toxin. Engineered mutants of HDC1 had smaller and less septate conidia and exhibited an approximately 50% reduction in total HDAC activity. Mutants were strongly reduced in virulence as a result of reduced penetration efficiency. Growth of hdc1 mutants in vitro was normal on glucose, slightly decreased on sucrose, and reduced by 30 to 73% on other simple and complex carbohydrates. Extracellular depolymerase activities and expression of the corresponding genes were downregulated in hdc1 mutant strains. Except for altered conidial morphology, the phenotypes of hdc1 mutants were similar to those of C. carbonum strains mutated in ccSNF1 encoding a protein kinase necessary for expression of glucose-repressed genes. These results show that HDC1 has multiple functions in a filamentous fungus and is required for full virulence of C. carbonum on maize.     

Gene organization and plasticity of the β-lactam genes in different filamentous fungi

The genes pcbAB, pcbC and penDE encoding enzymes that catalyze the three steps of the penicillin biosynthesis have been cloned from Penicillium chrysogenum and Aspergillus nidulans. They are located in a cluster in Penicillium chrysogenum, Penicillium notatum, Aspergillus nidulans and Penicillium nalgiovense. The three genes are clustered in chromosome I (10.4 Mb) of P. chrysogenum, in chromosome II of P. notatum (9.6 Mb) and in chromosome VI (3.0 Mb) of A. nidulans. The cluster of the penicillin biosynthetic genes is amplified in strains with high level of antibiotic production. About five to six copies of the cluster are present in the AS-P-78 strain and 11 to 14 copies in the E1 strain (an industrial isolate), whereas only one copy is present in the wild type (NRRL 1951) strain and in the low producer Wis 54-1255 strain. The amplified region in strains AS-P-78 and E1 is arranged in tandem repeats of 106.5 or 57.6-kb units, respectively. In Acremonium chrysogenum the genes involved in cephalosporin biosynthesis are separated in at least two clusters. The pcbAB and pcbC genes are linked in the so-called early cluster of genes involved in the cephalosporin biosynthesis. The late cluster, which includes the cefEF and cefG genes, is involved in the last steps of cephalosporin biosynthesis. The early cluster was located in chromosome VII (4.6 Mb) in the C10 strain and the late cluster in chromosome I (2.2 Mb). Both clusters are present in a single copy in the A. chrysogenum genome, in the wild-type and in the high cephalosporin-producing C10 strains.     

Cloning and targeted gene disruption of EXG1, encoding exo-beta 1, 3-glucanase, in the phytopathogenic fungus Cochliobolus carbonum.

The phytopathogenic fungus Cochliobolus carbonum produces an extracellular enzyme capable of degrading beta 1,3-glucan in an exolytic manner. On the basis of partial amino acid sequences of the purified enzyme, two degenerate oligonucleotides were synthesized and used as PCR primers to amplify a 1.1-kb fragment of corresponding genomic DNA. The PCR product was used to isolate the genomic copy of the gene, called EXG1. Partial sequencing of the genomic DNA confirmed that the PCR product corresponded to EXG1. A strain of the fungus specifically mutated in the EXG1 gene was constructed by homologous integration of an internal fragment of EXG1. In the mutant, enzymatic activity and the corresponding peak of UV absorption during high-pressure liquid chromatography purification were reduced by at least 98%. However, crude culture filtrates of the mutant retained 44% of the wild-type beta 1,3-glucanase activity. This residual activity was due to two additional activities which were chromatographically separable from the product of EXG1 and which were coeluted with beta 1,3-beta 1,4-glucanase activity. Growth of the EXG1 mutant was normal on sucrose and oat bran but was reduced by 65% on pure beta 1,3-glucan. The EXG1 mutant was still pathogenic to maize.     

Genome plasticity in Streptomyces: identification of 1 Mb TIRs in the S. coelicolor A3(2) chromosome

The chromosomes of several widely used laboratory derivatives of Streptomyces coelicolor A3(2) were found to have 1.06 Mb inverted repeat sequences at their termini (i.e. long-terminal inverted repeats; L-TIRs), which are 50 times the length of the 22 kb TIRs of the sequenced S. coelicolor strain M145. The L-TIRs include 1005 annotated genes and increase the overall chromosome size to 9.7 Mb. The 1.06 Mb L-TIRs are the longest reported thus far for an actinomycete, and are proposed to represent the chromosomal state of the original soil isolate of S. coelicolor A3(2). S. coelicolor A3(2), M600 and J1501 possess L-TIRs, whereas approximately half the examined early mutants of A3(2) generated by ultraviolet (UV) or X-ray mutagenesis have truncated their TIRs to the 22 kb length. UV radiation was found to stimulate L-TIR truncation. Two copies of a transposase gene (SCO0020) flank 1.04 Mb of DNA in the right L-TIR, and recombination between them appears to generate strains containing short TIRs. This TIR reduction mechanism may represent a general strategy by which transposable elements can modulate the structure of chromosome ends. The presence of L-TIRs in certain S. coelicolor strains represents a major chromosomal alteration in strains previously thought to be genetically similar.     

Histone Hyperacetylation in Maize in Response to Treatment with HC-Toxin or Infection by the Filamentous Fungus Cochliobolus carbonum

HC-toxin, the host-selective toxin produced by the filamentous fungus Cochliobolus carbonum, inhibits maize (Zea mays L.) histone deacetylases (HDs) in vitro. Here we show that HDs are also inhibited by HC-toxin in vivo, as demonstrated by the accumulation of hyperacetylated forms of the core (nucleosomal) histones H3.1, H3.2, H3.3, and H4 in both maize embryos and tissue cultures. Hyperacetylation of H4 and all isoforms of H3 in tissue cultures of inbred Pr (genotype hm/hm) occurred at 10 ng/mL (23 nM). The effect was host-selective; acetylation of histones in the near isogenic inbred Pr1 (genotype Hm/Hm) did not occur in tissue cultures or embryos treated with 0.2 [mu]g/mL or 10 [mu]g/mL HC-toxin, respectively. Hyperacetylation of histone H4 in embryos of Pr1 began to occur at 50 [mu]g/mL. HC-toxin, and 200 [mu]g/mL HC-toxin caused equal hyperacetylation in Pr and Pr1 embryos. Hyperacetylated core histones, especially of the isoforms of histone H3, accumulated in leaves of inbred Pr, but not Pr1, after infection by toxin-producing strains of C. carbonum. Accumulation of hyperacetylated histones began at 24 h after inoculation, before the development of visible disease symptoms. Hyperacetylation of H2A or H2B histones were not detected in any of the studies. The results are consistent with HD being a primary site of action of HC-toxin.     

Chromosomal polymorphism of Acremonium chrysogenum strains producing cephalosporin C

Using pulse electrophoresis in controlled homogenous electric field we performed molecular karyotyping of cephalosporin C-producing industrial and laboratory strains of Acremonium chrysogenum. Differences in size of several chromosomes of high-producing strain CB26/8 compared to the wild-type strain ATCC 11550 were revealed. It was shown that chromosomal polymorphism in the high-producing strain was not associated with alteration of localization and copy number of cephalosporin C (CPC) biosynthesis and transport genes. A cluster of ??early?? CPC biosynthesis genes is located on chromosome VI (4.4 Mb); a cluster of the ??late genes??, on chromosome II (2.3 Mb). Both clusters are presented as a single copy per A. chrysogenum genome in the wild-type and in CB26/8 high-producing strains. Based on comparative analysis of laboratory and industrial CPC producers, a karyotype scheme for A. chrysogenum strains of various origins was designed.     

Molecular characterization of the afl-1 locus in Aspergillus flavus.

An unusual mutation at the afl-1 locus, affecting aflatoxin biosynthesis in Aspergillus flavus 649, was investigated. The inability of strain 649 to produce aflatoxin was found to be the result of a large (greater than 60 kb) deletion that included a cluster of aflatoxin biosynthesis genes. Diploids formed by parasexual crosses between strain 649 and the aflatoxigenic strain 86 did not produce aflatoxin, indicating the dominant nature of the afl-1 mutation in strain 649. In metabolite feeding experiments, the diploids did not convert three intermediates in the aflatoxin pathway to aflatoxin. Northern (RNA blot) analysis of the diploids grown in medium conducive for aflatoxin production indicated that the aflatoxin pathway genes nor1, ver1, and omt1 were not expressed; however, there was low-level expression of the regulatory gene aflR. Pulsed-field electrophoresis gels indicated a larger (6 Mb) chromosome in strain 649 than the apparently homologous (4.9 Mb) chromosome in strain 86. The larger chromosome in strain 649 suggests that a rearrangement occurred in addition to the deletion. From these data, we proposed that a trans-sensing mechanism in diploids is responsible for the dominant phenotype associated with the afl-1 locus in strain 649. Such a mechanism is known in Drosophila melanogaster but has not been described for fungi.     

An inhibitor-resistant histone deacetylase in the plant pathogenic fungus Cochliobolus carbonum.

We have partially purified and characterized histone deacetylases of the plant pathogenic fungus Cochliobolus carbonum. Depending on growth conditions, this fungus produces HC-toxin, a specific histone deacetylase inhibitor. Purified enzymes were analyzed by immunoblotting, by immunoprecipitation, and for toxin sensitivity. The results demonstrate the existence of at least two distinct histone deacetylase activities. A high molecular weight complex (430,000) is sensitive to HC-toxin and trichostatin A and shows immunoreactivity with an antibody against Cochliobolus HDC2, an enzyme homologous to yeast RPD3. The second activity, a 60,000 molecular weight protein, which is resistant even to high concentrations of well-known deacetylase inhibitors, such as HC-toxin and trichostatin A, is not recognized by antibodies against Cochliobolus HDC1 (homologous to yeast HOS2) or HDC2 and represents a different and/or modified histone deacetylase which is enzymatically active in its monomeric form. This enzyme activity is not present in the related filamentous fungus Aspergillus nidulans. Furthermore, in vivo treatment of Cochliobolus mycelia with trichostatin A and analysis of HDACs during the transition from non-toxin-producing to toxin-producing stages support an HC-toxin-dependent enzyme activity profile.     

Chromosome of the enterohemorrhagic Escherichia coli O157:H7; comparative analysis with K-12 MG1655 revealed the acquisition of a large amount of foreign DNAs.

A complete Xba I and Bln I cleavage map was constructed for the chromosome of an enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain isolated from an outbreak in Sakai City, Japan, in 1996. A comparative chromosome analysis with E. coli K-12 strain MG1655 was made. The EHEC chromosome was approximately 5600 kb in length, 1 Mb larger than that of MG1655. Despite the marked difference in chromosome length, the location and direction of seven rRNA operons of the EHEC strain were similar to those for MG1655. Overall organization of genes common in both strains is also highly conserved. Chromosome expansion was observed throughout the EHEC chromosome, albeit in an uneven manner. A large portion of the chromosome enlargement was observed in the region surrounding the replication terminus, particularly in a segment containing the terA locus. Sample sequencing of 3627 random shotgun clones suggested the presence of approximately 1550 kb strain-specific DNAs on the EHEC chromosome, most of which are likely to be of foreign origin.     

Construction of a Not I restriction map of the fission yeast Schizosaccharomyces pombe genome.

Pulsed field gel electrophoresis and large DNA technology were used to construct a Not I restriction map of the entire genome of the fission yeast Schizosaccharomyces pombe. There are 14 detectable Not I sites in S. pombe 972h: 9 sites on chromosome I and 5 sites on chromosome II, while no Not I sites were found on chromosome III. The 17 fragments (including intact chromosome III) generated by Not I digestion were resolved by PFG electrophoresis. These fragments ranged in size from 4.5 kb to approximately 3.5 Mb. Various strategies were applied in determining, efficiently, the order of the fragments on the chromosomes. The genomic size measured by adding all the fragments together is about 14 Mb and the sizes of the three chromosomes are I, 5.7 Mb, II, 4.6 to 4.7 Mb, and III, 3.5 Mb. These are generally somewhat smaller than estimated previously.