BackgroundThe plant pathogenic basidiomycete Sclerotium rolfsii produces the industrially exploited exopolysaccharide scleroglucan, a polymer that consists of (1 → 3)-β-linked glucose with a (1 → 6)-β-glycosyl branch on every third unit. Although the physicochemical properties of scleroglucan are well understood, almost nothing is known about the genetics of scleroglucan biosynthesis. Similarly, the biosynthetic pathway of oxalate, the main by-product during scleroglucan production, has not been elucidated yet. In order to provide a basis for genetic and metabolic engineering approaches, we studied scleroglucan and oxalate biosynthesis in S. rolfsii using different transcriptomic approaches.ResultsTwo S. rolfsii transcriptomes obtained from scleroglucan-producing and scleroglucan-nonproducing conditions were pooled and sequenced using the 454 pyrosequencing technique yielding ~350,000 reads. These could be assembled into 21,937 contigs and 171,833 singletons, for which 6,951 had significant matches in public protein data bases. Sequence data were used to obtain first insights into the genomics of scleroglucan and oxalate production and to predict putative proteins involved in the synthesis of both metabolites. Using comparative transcriptomics, namely Agilent microarray hybridization and suppression subtractive hybridization, we identified ~800 unigenes which are differently expressed under scleroglucan-producing and non-producing conditions. From these, candidate genes were identified which could represent potential leads for targeted modification of the S. rolfsii metabolism for increased scleroglucan yields.ConclusionsThe results presented in this paper provide for the first time genomic and transcriptomic data about S. rolfsii and demonstrate the power and usefulness of combined transcriptome sequencing and comparative microarray analysis. The data obtained allowed us to predict the biosynthetic pathways of scleroglucan and oxalate synthesis and to identify important genes putatively involved in determining scleroglucan yields. Moreover, our data establish the first sequence database for S. rolfsii, which allows research into other biological processes of S. rolfsii, such as host-pathogen interaction.
The afp gene encoding the antifungal protein (AFP) of Aspergillus giganteus has a prototypical alkaline gene expression pattern, which suggests that the gene might be under the control of the ambient pH-dependent zinc-finger transcription factor PacC. This notion is corroborated by the presence in the upstream region of afp of two putative PacC binding sites, afpP1 and afpP2, which are specifically recognised by the PacC protein of A. nidulans in vitro. However, in this report we provide several lines of evidence to show that pH-dependent up-regulation of afp is not mediated by transcriptional activation through PacC. (1) The temporal expression pattern of the A. giganteus pacC gene does not parallel the accumulation of the afp mRNA during cultivation. (2) Inactivation of afpP1 and afpP2 did not reduce promoter activity under alkaline conditions, as determined from the relative wild-type and mutant afp::lacZ reporter activities in A. nidulans. (3) Reporter activities in acidity- and alkalinity-mimicking mutant strains are inconsistent with a positive role for PacC in afp expression. (4) In A. giganteus, the pH-dependent increase in afp mRNA and AFP levels can be completely prevented by the calcineurin inhibitor FK506, suggesting that the calcineurin signalling pathway might control the in vivo activation of the afp promoter by alkaline pH.
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