Molecular cloning and characterization of cel2 from the fungus Cochlibolus carbonum

A new cellulase gene, cel2, from the filamentous fungus Cochliobolus carbonum was cloned by using egl-1 of Trichoderma reesei as a heterologous probe. DNA blot analysis of cel2 showed that this gene is present as a single copy. The gene contains one 49-bp- intron. cel2 encodes a predicted protein (Cel2p) of 423 amino acids with a molecular mass of 45.8 kDa. The predicted pI is 4.96. It shows similarity to other endoglucanases from various fungi. From the comparison with other cellulase genes, cel2 belongs to family 7 of glucohydrolases. cel2 is located on a 2.5-Mb chromosome in C. carbonum and its expression is repressed by sucrose. A cel2 mutant of C. carbonum was created by transformation-mediated gene disruption. The pathogenicity of the mutant was indistinguishable from the wild type, indicating that cel2 by itself is not important for pathogenicity.     

Cloning, disruption, and expression of two endo-beta 1, 4-xylanase genes, XYL2 and XYL3, from Cochliobolus carbonum.

In culture, the filamentous fungus Cochliobolus carbonum, a pathogen of maize, makes three cationic xylanases, XYL1, which encodes the major endoxylanase (Xyl1), was earlier cloned and shown by gene disruption to encode the first and second peaks of xylanase activity (P. C. Apel, D. G. Panaccione, F. R. Holden, and J. D. Walton, Mol. Plant-Microbe Interact. 6:467-473, 1993). Two additional xylanase genes, XYL2 and XYL3, have now been cloned from C. carbonum. XYL2 and XYL3 are predicted to encode 22-kDa family G xylanases similar to Xyl1. Xyl2 and Xyl3 are 60% and 42% identical, respectively, to Xyl1, and Xyl2 and Xyl3 are 39% identical. XYL1 and XYL2 but not XYL3 mRNAs are present in C. carbonum grown in culture, and XYL1 and XYL3 but not XYL2 mRNAs are present in infected plants. Transformation-mediated gene disruption was used to construct strains mutated in XYL1, XYL2, and XYL3. Xyl1 accounts for most of the total xylanase activity in culture, and disruption of XYL2 or XYL3 does not result in the further loss of any xylanase activity. In particular, the third peak of cationic xylanase activity is still present in a xyl1 xyl2 xyl3 triple mutant, and therefore this xylanase must be encoded by yet a fourth xylanase gene. A minor protein of 22 kDa that can be detected immunologically in the xyl1 mutant disappears in the xyl2 mutant and is therefore proposed to be the product of XYL2. The single xylanase mutants were crossed with each other to obtain multiple xylanase disruptions within the same strain. Strains disrupted in combinations of two and in all three xylanases were obtained. The triple mutant grows at the same rate as the wild type on xylan and on maize cell walls. The triple mutant is still fully pathogenic on maize with regard to lesion size, morphology, and rate of lesion development.     

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.     

Endopolygalacturonase is not required for pathogenicity of Cochliobolus carbonum on maize.

A gene (PGN1) encoding extracellular endopolygalacturonase was isolated from the fungal maize pathogen Cochliobolus carbonum race 1. A probe was synthesized by polymerase chain reaction using oligonucleotides based on the endopolygalacturonase amino acid sequence. Genomic and cDNA copies of the gene were isolated and sequenced. The corresponding mRNA was present in C. carbonum grown on pectin but not on sucrose as carbon source. The single copy of PGN1 in C. carbonum was disrupted by homologous integration of a plasmid containing an internal fragment of the gene. Polygalacturonase activity in one transformant chosen for further analysis was 10% or 35% of the wild-type activity based on viscometric or reducing sugar assays, respectively. End product analysis indicated that the residual activity in the mutant was due to an exopolygalacturonase. Pathogenicity on maize of the mutant lacking endopolygalacturonase activity was qualitatively indistinguishable from the wild-type strain, indicating that in this disease interaction endopolygalacturonase is not required. Either pectin degradation is not critical to this interaction or exopolygalacturonase alone is sufficient.     

Analysis of a Clostridium josui cellulase gene cluster containing the man5A gene and characterization of recombinant Man5A

A cellulase gene cluster of Clostridium josui was sequenced, and was found to encode 11 proteins responsible for cellulosome (cellulolytic complex) formation, viz., cipA, cel48A, cel8A, cel9A, cel9B, orfX, cel9C, cel9D, man5A, cel9E, and cel5B, in order from the upstream side. All the predicted enzymes had a dockerin module, suggesting that these proteins are members of the C. josui cellulosome. Among these genes, the man5A gene encoding β-mannanase was expressed in Escherichia coli and the recombinant enzyme (rMan5A) was characterized. rMan5A showed strong activity toward carob galactomannan and low activity toward guar gum, suggesting that it prefers non-galactosylated mannan to galactomannan. This enzyme hydrolyzed ivory nut mannan to produce mainly mannotriose and larger mannooligosaccharides, and was not active toward mannotriose. An antiserum raised against the recombinant enzyme detected Man5A in the culture supernatants of C. josui, which was grown on either ball-milled cellulose or glucose as a carbon source.     

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.     

Cloning of the cel8Y gene from Pectobacterium chrysanthemi PY35 and its comparison to cel genes of soft-rot Pectobacterium

The phytopathogenic Pectobacterium chrysanthemi (Pch) PY35 secretes multiple isozymes of plant cell wall degrading enzyme cellulases. We cloned a second cel gene that encodes cellulase in Pch PY35. The inserted 2 kb fragment was subcloned in order to geneate pPY710 (cel8Y). The structural organization of the cel8Y gene consists of an open reading frame (ORF) of 999 bp that encodes 332 amino acid residues with a signal peptide of 23 amino acids. The predicted amino acid sequence of Cel8Y was very similar to that of Cellulomonas uda, but completely different from that of the Cel5Z of Pch PY35. It belonged to the glycoside hydrolase family 8, based on amino acid sequence similarities in contrast to Cel5Z of Pch PY35, which was confirmed as family 5. Cel8Y was not closely related to the known cellulases of Pectobacterium. It had the conserved region of the glycoside hydrolase family 8, ASDGDVLIAWALLKAGNKW. The apparent molecular mass of the Cel8Y protein was calculated to be approximately 34 kDa by a carboxymethylcellulosesodium dodecyl sulfate-polyacrylamide gel electrophoresis (CMC-SDS-PAGE). The Cel8Y had a calculated pl of 6.49. It was optimally active at pH 7 with an approximate optimal temperature around 40 degrees C. The cellulase activity of Cel8Y was lower than that of Cel5Z.     

Cloning and DNA sequence of the gene coding for Clostridium thermocellum cellulase Ss (CelS), a major cellulosome component.

Clostridium thermocellum ATCC 27405 produces an extracellular cellulase system capable of hydrolyzing crystalline cellulose. The enzyme system involves a multicomponent protein aggregate (the cellulosome) with a total molecular weight in the millions, impeding mechanistic studies. However, two major components of the aggregate, SS (M(r) = 82,000) and SL (M(r) = 250,000), which act synergistically to hydrolyze crystalline cellulose, have been identified (J. H. D. Wu, W. H. Orme-Johnson, and A. L. Demain, Biochemistry 27:1703-1709, 1988). To further study this synergism, we cloned and sequenced the gene (celS) coding for the SS (CelS) protein by using a degenerate, inosine-containing oligonucleotide probe whose sequence was derived from the N-terminal amino acid sequence of the CelS protein. The open reading frame of celS consisted of 2,241 bp encoding 741 amino acid residues. It encoded the N-terminal amino acid sequence and two internal peptide sequences determined for the native CelS protein. A putative ribosome binding site was identified at the 5' end of the gene. A putative signal peptide of 27 amino acid residues was adjacent to the N terminus of the CelS protein. The predicted molecular weight of the secreted protein was 80,670. The celS gene contained a conserved reiterated sequence encoding 24 amino acid residues found in proteins encoded by many other clostridial cel or xyn genes. A palindromic structure was found downstream from the open reading frame. The celS gene is unique among the known cel genes of C. thermocellum. However, it is highly homologous to the partial open reading frame found in C. cellulolyticum and in Caldocellum saccharolyticum, indicating that these genes belong to a new family of cel genes.     

Identification and DNA sequence of a pathogenicity gene of Xanthomonas campestris pv. campestris

A region of Xanthomonas campestris pv. campestris DNA containing at least two pathogenicity genes was identified. Mutants in one gene were clearly reduced in pathogenicity while mutants in the other were only moderately reduced. Both classes of mutants were prototrophic and motile, and had wild-type levels of extracellular enzymes and extracellular polysaccharide. They also grew in vitro and in planta at the same rate as the wild type. Experiments involving one of the clear pathogenicity mutants indicated that the recovery of mutant cells from turnip seedlings 24 hr after inoculation was lower than for the wild type. This may be due to cell death as a result of action by some preformed or induced plant factor. From DNA sequencing an open reading frame was identified that encompassed the site of the mutations giving a clear reduction in pathogenicity. The predicted protein sequence had no homology with other proteins in the computer data base.     

Gene Cloning of Endoglucanase Cel5A from Cellulose-Degrading <Emphasis Type="Italic">Paenibacillus xylanilyticus</Emphasis> KJ-03 and Purification and Characterization of the Recombinant Enzyme

The bacterial strain Paenibacillus xylanilyticus KJ-03 was isolated from a sample of soil used for cultivating Amorphophallus konjac. The cellulase gene, cel5A was cloned using fosmid library and expressed in Escherichia coli BL21 (trxB). The cel5A gene consists of a 1,743 bp open reading frame and encodes 581 amino acids of a protein. Cel5A contains N-terminal signal peptide, a catalytic domain of glycosyl hydrolase family 5, and DUF291 domain with unknown function. The recombinant cellulase was purified by Ni-affinity chromatography. The cellulase activity of Cel5A was detected in clear band with a molecular weight of 64 kDa by zymogram active staining. The maximum activity of the purified enzyme was displayed at a temperature of 40 °C and pH 6.0 when carboxymethyl cellulose was used as a substrate. It has 44% of its maximum activity at 70 °C and retained 66% of its original activity at 45 °C for 1 h. The purified cellulase hydrolyzed avicel, CMC, filter paper, xylan, and 4-methylumbelliferyl β-d-cellobiose, but no activity was detected against p-nitrophenyl β-d-glucoside. The end products of the hydrolysis of cellotetraose and cellopentaose by Cel5A were detected by thin layer chromatography, while enzyme did not hydrolyze cellobiose and cellotriose.     

Polymorphic Chromosomes Bearing the Tox2 Locus in Cochliobolus carbonum Behave as Homologs during Meiosis

The HTS1 gene in the Tox2 locus of the fungal pathogen Cochliobolus carbonum race 1 is required for synthesis of a host-selective phytotoxin and for increased virulence on susceptible genotypes of maize. The locus is present in race 1 isolates but absent from isolates of the other races, which do not produce the toxin. By pulsed-field gel electrophoresis and Southern analysis with HTS1 sequences and chromosome-specific markers, the HTS1 gene was detected on a 4-Mb chromosome in one group of isolates and on a 2.3-Mb chromosome in another group, which lacked the 4-Mb chromosome. A chromosome-specific marker from C. heterostrophus hybridized to a 2.3-Mb chromosome in non-toxin-producing isolates and in toxin-producing isolates, including those with a 4-Mb chromosome. A marker from C. carbonum hybridized to the 4-Mb chromosome, but in isolates lacking the 4-Mb chromosome, this marker hybridized to a smaller, 2.0-Mb chromosome. Thus, the Tox2 locus is on different chromosomes in different groups of race 1 isolates. Single ascospore progeny from crosses between isolates having HTS1 on different chromosomes were analyzed for toxin-producing ability, virulence, and the presence and chromosomal location of HTS1. All progeny produced HC toxin in culture, incited race 1-type lesions on susceptible maize genotypes, and contained HTS1 sequences, as determined by PCR amplification with gene-specific primers. Analysis of the chromosomal complements of several progeny indicated that they all had only one Tox2-containing chromosome. Thus, despite their differences in size, these chromosomes behave as homologs during meiosis and may have arisen by a translocation.