SummaryPenicillium chrysogenum is the main industrial producer of the β-lactam antibiotic penicillin, the most commonly used drug in the treatment of bacterial infections. Recently, a functional MAT1-1 locus encoding the α-box transcription factor MAT1-1-1 was discovered to control sexual development in P. chrysogenum. As only little was known from any organism about the regulatory functions mediated by MAT1-1-1, we applied chromatin immunoprecipitation combined with next-generation sequencing (ChIPseq) to gain new insights into the factors that influence MAT1-1-1 functions on a molecular level and its role in genome-wide transcriptional regulatory networks. Most importantly, our data provide evidence for mating-type transcription factor functions that reach far beyond their previously understood role in sexual development. These new roles include regulation of hyphal morphology, asexual development, as well as amino acid, iron, and secondary metabolism. Furthermore, in vitro DNA-protein binding studies and downstream analysis in yeast and P. chrysogenum enabled the identification of a MAT1-1-1 DNAbinding motif, which is highly conserved among euascomycetes. Our studies pave the way to a more general understanding of these master switches for development and metabolism in all fungi, and open up new options for optimization of fungal high production strains.
SummaryThe filamentous fungus Sordaria macrospora is a model system to study multicellular development during fruiting body formation. Previously, we demonstrated that this major process in the sexual life cycle is controlled by the Zn(II) 2 Cys 6 zinc cluster transcription factor PRO1. Here, we further investigated the genome-wide regulatory network controlled by PRO1 by employing chromatin immunoprecipitation combined with next-generation sequencing (ChIP-seq) to identify binding sites for PRO1. We identified several target regions that occur in the promoter regions of genes encoding components of diverse signaling pathways. Furthermore, we identified a conserved DNA-binding motif that is bound specifically by PRO1 in vitro. In addition, PRO1 controls in vivo the expression of a DsRed reporter gene under the control of the esdC target gene promoter. Our ChIP-seq data suggest that PRO1 also controls target genes previously shown to be involved in regulating the pathways controlling cell wall integrity, NADPH oxidase and pheromone signaling. Our data point to PRO1 acting as a master regulator of genes for signaling components that comprise a developmental cascade controlling fruiting body formation.
BackgroundMulti-copy gene integration into microbial genomes is a conventional tool for obtaining improved gene expression. For Penicillium chrysogenum, the fungal producer of the beta-lactam antibiotic penicillin, many production strains carry multiple copies of the penicillin biosynthesis gene cluster. This discovery led to the generally accepted view that high penicillin titers are the result of multiple copies of penicillin genes. Here we investigated strain P2niaD18, a production line that carries only two copies of the penicillin gene cluster.ResultsWe performed pulsed-field gel electrophoresis (PFGE), quantitative qRT-PCR, and penicillin bioassays to investigate production, deletion and overexpression strains generated in the P. chrysogenum P2niaD18 background, in order to determine the copy number of the penicillin biosynthesis gene cluster, and study the expression of one penicillin biosynthesis gene, and the penicillin titer. Analysis of production and recombinant strain showed that the enhanced penicillin titer did not depend on the copy number of the penicillin gene cluster. Our assumption was strengthened by results with a penicillin null strain lacking pcbC encoding isopenicillin N synthase. Reintroduction of one or two copies of the cluster into the pcbC deletion strain restored transcriptional high expression of the pcbC gene, but recombinant strains showed no significantly different penicillin titer compared to parental strains.ConclusionsHere we present a molecular genetic analysis of production and recombinant strains in the P2niaD18 background carrying different copy numbers of the penicillin biosynthesis gene cluster. Our analysis shows that the enhanced penicillin titer does not strictly depend on the copy number of the cluster. Based on these overall findings, we hypothesize that instead, complex regulatory mechanisms are prominently implicated in increased penicillin biosynthesis in production strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-017-0335-8) contains supplementary material, which is available to authorized users.
Staphylococcus lugdunensis is an important human pathogen that causes infectious diseases similar to those caused by Staphylococcus aureus. In contrast to S. aureus, only a very few pathogenicity factors of S. lugdunensis have been characterized. Notably, a genetic manipulation of S. lugdunensis has not yet been described. Ours is the first report where transformation of three different plasmids (pBT2, pRB473, and pT181) into S. lugdunensis and a directed genetic manipulation of S. lugdunensis are described. We constructed fbl knockout mutants from three different strains of S. lugdunensis to show that at least in these strains, the fibrinogen binding is exclusively mediated by Fbl.
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