Chivosazol A

Microorganisms continue to be a source of novel, bioactive natural products for the treatment of human diseases. Notable among them are the myxobacteria, with some 50% of metabolites isolated from strains of a single species, Sorangium cellulosum. As native production in myxobacteria is often low, however, research has begun to address the regulatory systems governing the pathways, with the aim of increasing fermentation titers. These efforts are significantly enabled by whole genome sequencing data. We previously identified ChiR as a positive regulator of chivosazol biosynthesis in the genome sequencing strain S. cellulosum So ce56, only the second regulatory function known from myxobacterial secondary metabolism. As So ce56 is known to produce two additional compounds, the mixed polyketide etnangien (Irschik et al., 2007; Menche et al., 2008), and the siderophore myxochelin (Schneiker et al., 2007), we set out to further exploit the genome data to discover additional regulators of secondary metabolite biosynthesis. Here we report a novel function for a member of the NtcA family of nitrogen-responsive transcriptional regulators, as a negative transcriptional regulator of chivosazol biosynthesis. NtcA is a promoter binding protein (PBP), which recognizes a conserved sequence within the chivosazol promoter. Inactivation of ntcA enhanced the production of chivosazol by 4-fold, but also increased the yield of etnangien by 3.5-fold. The ammonia-induced repression of biosynthesis observed in wild type So ce56 was significantly attenuated in a ntcA mutant. Taken together, these data suggest that inhibition of chivosazol biosynthesis by environmental nitrogen is mediated, at least in part, by the NtcA protein. Our results also reinforce the idea that genomics-guided engineering of regulatory pathways is a viable strategy for improving metabolite yields through fermentation.

Actin is a protein of central importance for many cellular key processes. It is regulated by local interactions with a large number of actin binding proteins (ABPs). Various compounds are known to either increase or decrease the polymerization dynamics of actin. However, no actin binding compound has been developed for clinical applications yet because of selectivity issues. We provide a crystal structure of the natural product chivosazole A (ChivoA) bound to actin and show that-in addition to inhibiting nucleation, polymerization, and severing of F-actin filaments-it selectively modulates binding of ABPs to G-actin: Although unphysiological actin dimers are induced by ChivoA, interaction with gelsolin, profilin, cofilin, and thymosin-β4 is inhibited. Moreover, ChivoA causes transcriptional effects differing from latrunculin B, an actin binder with a different binding site. Our data show that ChivoA and related compounds could serve as scaffolds for the development of actin binding molecules selectively targeting specific actin functions.

High content microscopy as a screening tool to identify bioactive agents has provided researchers with the ability to characterise biological activities at the level of single cells. Here, we describe the development and the application of a high content screening assay for the identification and characterisation of cytostatic bioactivities from Myxobacteria extracts. In an automated microscopy assay Sf9 insect cells were visualised utilising the stains bisbenzimide Hoechst 33342, calcein AM, and propidium iodide. Imaging data were processed by the ScanR Analysis-software to determine the ploidy and vitality of each cell and to quantify cell populations. More than 98% of the Sf9 cells were viable and the culture consisted of diploid ( approximately 30%), tetraploid ( approximately 60%), polyploidic (<10%) and apoptotic (<5%) cells. Treatment with the reference substances blasticidin, colchicine, paclitaxel, and cytochalasin D induced changes in ploidy and vitality, which were characteristic for the respective bioactive substance. Furthermore, crude extracts from the chivosazole producing Myxobacterium Sorangium cellulosum So ce56 induced an increase of polyploid cells and a decrease in total cell count, while a mutant producing nearly no chivosazole triggered none of these effects. Purified chivosazole induced the same effects as the wild type extract. Similar effects have been observed for the reference compound cytochalasin D. On the basis of this assay, crude extracts of ten different Myxobacteria cultures were screened. Three extracts exhibited strong cytotoxic activities, further five extracts induced weak changes in the ploidy distribution, and two extracts showed no detectable effect within the assay. Therefore, this robust assay provides the ability to discover and characterise cytotoxic and cytostatic bioactivities in crude bacterial extracts.