The development of genetic and molecular markers to register and commercialize Penicillium rubens (formerly Penicillium oxalicum) strain 212 as a biocontrol agent.

Penicillium oxalicum strain 212 (PO212) is an effective biocontrol agent (BCA) against a large number of economically important fungal plant pathogens. For successful registration as a BCA in Europe, PO212 must be accurately identified. In this report, we describe the use of classical genetic and molecular markers to characterize and identify PO212 in order to understand its ecological role in the environment or host. We successfully generated pyrimidine (pyr-) auxotrophic mutants. In addition we also designed specific oligonucleotides for the pyrF gene at their untranslated regions for rapid and reliable identification and classification of strains of P. oxalicum and P. rubens, formerly P. chrysogenum. Using these DNA-based technologies, we found that PO212 is a strain of P. rubens, and is not a strain of P. oxalicum. This work presents PO212 as the unique P. rubens strain to be described as a BCA and the information contained here serves for its registration and commercialization in Europe.

Assessment of drug candidates for broad-spectrum antiviral therapy targeting cellular pyrimidine biosynthesis.

Currently available antiviral drugs frequently induce side-effects or selection of drug-resistant viruses. We describe a novel antiviral principle based on targeting the cellular enzyme dihydroorotate dehydrogenase (DHODH). In silico drug design and biochemical evaluation identified Compound 1 (Cmp1) as a selective inhibitor of human DHODH in vitro (IC50 1.5±0.2nM). Crystallization data specified the mode of drug-target interaction. Importantly, Cmp1 displayed a very potent antiviral activity that could be reversed by co-application of uridine or other pyrimidine precursors, underlining the postulated DHODH-directed mode of activity. Human and animal cytomegaloviruses as well as adenoviruses showed strong sensitivity towards Cmp1 in cell culture-based infection systems with IC50 values in the low micromolar to nanomolar range. Particularly, broad inhibitory activity was demonstrated for various types of laboratory and clinically relevant adenoviruses. For replication of human cytomegalovirus in primary fibroblasts, antiviral mode of activity was attributed to the early stage of gene expression. A mouse in vivo model proved reduced replication of murine cytomegalovirus in various organs upon Cmp1 treatment. These findings suggested Cmp1 as drug candidate and validated DHODH as a promising cellular target for antiviral therapy.

The evolution and structural anatomy of the small molecule metabolic pathways in Escherichia coli.

The 106 small molecule metabolic (SMM) pathways in Escherichia coli are formed by the protein products of 581 genes. We can define 722 domains, nearly all of which are homologous to proteins of known structure, that form all or part of 510 of these proteins. This information allows us to answer general questions on the structural anatomy of the SMM pathway proteins and to trace family relationships and recruitment events within and across pathways. Half the gene products contain a single domain and half are formed by combinations of between two and six domains. The 722 domains belong to one of 213 families that have between one and 51 members. Family members usually conserve their catalytic or cofactor binding properties; substrate recognition is rarely conserved. Of the 213 families, members of only a quarter occur in isolation, i.e. they form single-domain proteins. Most members of the other families combine with domains from just one or two other families and a few more versatile families can combine with several different partners. Excluding isoenzymes, more than twice as many homologues are distributed across pathways as within pathways. However, serial recruitment, with two consecutive enzymes both being recruited to another pathway, is rare and recruitment of three consecutive enzymes is not observed. Only eight of the 106 pathways have a high number of homologues. Homology between consecutive pairs of enzymes with conservation of the main substrate-binding site but change in catalytic mechanism (which would support a simple model of retrograde pathway evolution) occurs only six times in the whole set of enzymes. Most of the domains that form SMM pathways have homologues in non-SMM pathways. Taken together, these results imply a pervasive "mosaic" model for the formation of protein repertoires and pathways.