PcchiB1, encoding a class V chitinase, is affected by PcVelA and PcLaeA, and is responsible for cell wall integrity in Penicillium chrysogenum

Abstract

Penicillin production in Penicillium chrysogenum is controlled by PcVelA and PcLaeA, two components of the regulatory velvet-like complex. Comparative microarray analysis with mutants lacking PcVelA or PcLaeA revealed a set of 62 common genes affected by the loss of both components. A downregulated gene in both knockout strains is PcchiB1, potentially encoding a class V chitinase. Under nutrient-depleted conditions, transcript levels of PcchiB1 are strongly upregulated, and the gene product contributes to more than 50 % of extracellular chitinase activity. Functional characterization by generating PcchiB1-disruption strains revealed that PcChiB1 is responsible for cell wall integrity and pellet formation in P. chrysogenum. Further, fluorescence microscopy with a DsRed-labelled chitinase suggests a cell wall association of the protein. An unexpected phenotype occurred when knockout strains were grown on media containing N-acetylglucosamine as the sole C and N source, where, in contrast to the recipient, a penicillin producer strain, the mutants and an ancestral strain show distinct mycelial growth. We discuss the relevance of this class V chitinase for morphology in an industrially important fungus. INTRODUCTIONPenicillium chrysogenum is the main industrial producer of the b-lactam antibiotic penicillin. In this filamentous ascomycete, developmental processes and secondary metabolism are tightly linked and controlled by diverse conserved regulators. Among these are PcVelA and PcLaeA, two global regulators, which have recently been shown to control penicillin biosynthesis as well as hyphal and conidiophore morphogenesis (Hoff et al., 2010a). Homologues of both proteins have been intensively studied in the model fungus Aspergillus nidulans (Calvo, 2008;Bok & Keller, 2004;Sarikaya Bayram et al., 2010). For example, it has been shown that both proteins, together with VelB, form the core of a regulatory heterotrimeric complex, which we refer to as the velvet complex (Bayram et al., 2008a). The general regulatory function of these proteins is highly conserved in diverse filamentous fungi (Calvo, 2008), and it has been shown that velvet-like complexes exist in other ascomycetes (Hoff et al., 2010a;Wiemann et al., 2010).Despite its importance for development and secondary metabolism, so far only a few high-throughput transcriptional analyses have been conducted with strains lacking components of the velvet complex. Cary et al. (2007) performed a microarray analysis with a DveA mutant of Aspergillus flavus to analyse the expression levels of 5002 genes. That study identified 136 differentially regulated genes in the disruption strain compared with the control strain, some of these representing known aflatoxin biosynthetic genes. In Aspergillus fumigatus, about 20-40 % of all secondary metabolite biosynthesis genes are negatively affected by the loss of laeA, as detected by whole-genome microarray analysis (Perrin et al., 2007).Using a PcvelA knockout strain, our previous microarray study to understand the expression profi...

Members of the Penicillium chrysogenum Velvet Complex Play Functionally Opposing Roles in the Regulation of Penicillin Biosynthesis and Conidiation

“…S4 in the supplemental material). Previously, we have also shown that in this mutant, the level of PcvosA transcript is decreased (45), whereas that of PclaeA is increased in a ⌬PcvelA mutant (26). We therefore concluded that PcLaeA acts as a positive and negative regulator of PcvosA and PcvelC transcription, respectively.…”

“…In P. chrysogenum, deletion of PclaeA led to an increase of PcvelC transcripts, resulting most likely in an increased amount of the repressor protein. Furthermore, a decrease of PcvosA expression was observed in another array analysis (45), suggesting that proper signaling through this protein might be disturbed. PcLaeA is known to be negatively controlled upstream by PcVelA (26); thus, deletion of both proteins seems to mediate conidiation to a downstream component, most probably PcVosA, in an unimpeded manner.…”

“…Microarray analysis was performed as described previously by Hoff et al (26). The microarray data used in this study are available from the GSE18585 series at the NCBI Gene Expression Omnibus (GEO) repository (26,45).…”

Sexual reproduction and mating-type–mediated strain development in the penicillin-producing fungus Penicillium chrysogenum

“…DNA-mediated transformation of P. chrysogenum to construct gene deletion and overexpression strains and further rescue of deletion strains (Fig. S2) were done in principal as described recently (49). Detailed information on strain constructions and functional analysis is provided in SI Materials and Methods and Tables S4 and S5.…”

The Taming of the Shrew - Controlling the Morphology of Filamentous Eukaryotic and Prokaryotic Microorganisms

“…The authors suggested a down-regulation of genes involved in chitin degradation by the deletion of PcvelA as a possible reason for the observed hyper-branching phenotype, pellet formation, and delayed fragmentation and autolysis. This possibility was confirmed by microarray analyses with a PcvelA knockout strain in which the gene PcchiB1, encoding for a class V chitinase, was down-regulated [149].…”