Immunity against NS3 protein of classical swine fever virus does not protect against lethal challenge infection.

Classical swine fever is a highly contagious disease of swine caused by classical swine fever virus, an OIE list A pathogen. In the European Union the virus has been eradicated from the domestic pig population and prophylactic immunization has been banned. Nevertheless, intervention immunizations using marker vaccines are one possibility to deal with reintroduced CSFV. At present, baculovirus-expressed E2 protein is used as such a marker vaccine. However, this vaccine cannot fully protect against viral spread; hence the use of another subunit, or of a combination of two or more subunits, might be beneficial. Therefore the immunological effects of nonstructural protein 3 (NS3) on the humoral as well as the cellular arms of the immune system were investigated. Although effectors of both sides of the immune system were stimulated by application of recombinant NS3 protein, no protection against lethal CSFV challenge could be achieved.

Biosynthetic gene cluster of the herbicide phosphinothricin tripeptide from Streptomyces viridochromogenes Tü494.

The antibiotic phosphinothricin tripeptide (PTT) consists of two molecules of L-alanine and one molecule of the unusual amino acid phosphinothricin (PT) which are nonribosomally combined. The bioactive compound PT has bactericidal, fungicidal, and herbicidal properties and possesses a C-P-C bond, which is very rare in natural compounds. Previously uncharacterized flanking and middle regions of the PTT biosynthetic gene cluster from Streptomyces viridochromogenes Tü494 were isolated and sequenced. The boundaries of the gene cluster were identified by gene inactivation studies. Sequence analysis and homology searches led to the completion of the gene cluster, which consists of 24 genes. Four of these were identified as undescribed genes coding for proteins that are probably involved in uncharacterized early steps of antibiotic biosynthesis or in providing precursors of PTT biosynthesis (phosphoenolpyruvate, acetyl-coenzyme A, or L-alanine). The involvement of the genes orfM and trs and of the regulatory gene prpA in PTT biosynthesis was analyzed by gene inactivation and overexpression, respectively. Insight into the regulation of PTT was gained by determining the transcriptional start sites of the pmi and prpA genes. A previously undescribed regulatory gene involved in morphological differentiation in streptomycetes was identified outside of the left boundary of the PTT biosynthetic gene cluster.

Tricarboxylic acid cycle aconitase activity during the life cycle of Streptomyces viridochromogenesTü494.

Previously, it was shown that inactivation of the tricarboxylic acid cycle aconitase gene acnA impairs the morphological and physiological differentiation of Streptomyces viridochromogenes Tü494, which produces the herbicide phosphinothricin tripeptide (PTT). In order to further characterize the role of the aconitase in the Streptomyces life cycle, aconitase activity was analyzed during growth of S. viridochromogenes in liquid culture. Two prominent maxima were measured in cell-free crude extracts. The first maximum was found at an early stage of growth, which is correlated with a decrease in pH when rapid glucose consumption is initiated. The second, lower maximum was detected at the beginning of the expression of the PTT-specific biosynthetic gene phsA,implying the onset of secondary metabolism. These results were confirmed by examining transcription of the acnA promoter in time-course experiments. The highest transcription rate was found during the early growth phases. In order to identify putative regulatory mechanisms, the transcriptional start site of the acnA transcript and subsequently the promoter were identified. Several putative, regulatory protein binding sites (e.g. regulators of oxygen stress or iron metabolism) were detected in the promoter region of acnA, which suggested complex regulation of acnA.