Expression of cmg1 , an Exo-β-1,3-Glucanase Gene from Coniothyrium minitans , Increases during Sclerotial Parasitism

Abstract

During sclerotial infection of Sclerotinia sclerotiorum the mycoparasite Coniothyrium minitans penetrates through the host cell wall, which contains ␤-1,3-glucan as its major component. A PCR-based strategy was used to clone a ␤-1,3-glucanase-encoding gene, designated cmg1, from a cDNA library of the fungus. The nucleotide and deduced amino acid sequences of this gene showed high levels of similarity to the sequences of other fungal exo-␤-1,3-glucanase genes. The calculated molecular mass of the deduced protein (without the predicted 24-amino-acid N-terminal secretion signal peptide) was 83,346 Da, and the estimated pI was 4.73. Saccharomyces cerevisiae INVSc1 expressing the cmg1 gene secreted a ϳ100-kDa ␤-1,3-glucanase enzyme (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) into the culture medium. N-terminal sequence analysis of the purified recombinant enzyme revealed that the secreted enzyme starts at Ala-32, seven amino acids downstream from the predicted signal peptidase cleavage site. The purified recombinant glucanase inhibited in vitro mycelial growth of S. sclerotiorum by 35 and 85% at concentrations of 300 and 600 g ml ؊1 , respectively. A single copy of the cmg1 gene is present in the genome of C. minitans. Northern analyses indicated increases in the transcript levels of cmg1 due to both carbon starvation and the presence of ground sclerotia of S. sclerotiorum; only slight repression was observed in the presence of 2% glucose. Expression of cmg1 increased during parasitic interaction with S. sclerotiorum.

Active Site and Laminarin Binding in Glycoside Hydrolase Family 55

“…Functional Survey of the GH55 Family-To date, the majority of studies on GH55 have been on fungal enzymes (19,20,(22)(23)(24)(25)(26)(27)61). With the exception of a single report on the cloning and sequence analysis of GluA from Arthrobacter sp.…”

“…With the exception of single report on the cloning and sequence analysis of GluA from Arthrobacter sp. NHB-1 (21), previous published work on GH55 has been on fungal members (19,20,(22)(23)(24)(25)(26)(27). This includes the GH55 from Phanerochaete chrysosporium, previously named PcLam55A (20), whose structure has also been determined.…”

Crystal structure and biological implications of a glycoside hydrolase family 55 β-1,3-glucanase from Chaetomium thermophilum

“…Recent genomic sequencing data have showed a number of β-1,3-glucanases genes from the GH55 family in fungi and bacteria and some β-1,3-glucanases of the GH55 family have been cloned, expressed and characterized [6][7][8][9][10][11][12][13][14]. However, the crystal structures of only two β-1,3-glucanases from the GH55 family have been reported so far, one (PcLam55A) from the fungus Phanerochaete chrysosporium [6] and the other (SacteLam55A) from the bacterial Streptomyces sp.…”

Disruption of the Coniothyrium minitans PIF1 DNA helicase gene impairs growth and capacity for sclerotial mycoparasitism

“…The cmg1 exo-b-1,3-glucanase gene was shown to be upregulated when the fungus was grown on media containing sclerotia as the sole carbon source (Giczey et al, 2001). Recently we have identified 251 unique transcripts of C. minitans upregulated during growth on sclerotia, and representing genes involved in diverse processes including signalling, detoxification and stress responses (Muthumeenakshi et al, 2007).…”