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There is increasing evidence that uncultivated bacterial symbionts are the true producers of numerous bioactive compounds isolated from marine sponges. The localization and heterologous expression of biosynthetic genes could clarify this issue and provide sustainable supplies for a wide range of pharmaceuticals. However, identification of genes in the usually highly complex symbiont communities remains a challenging task. For polyketides, one of the most important groups of sponge-derived drug candidates, we have developed a general strategy that allows one to rapidly access biosynthetic gene clusters based on chemical moieties. Using this method, we targeted polyketide synthase genes from two different sponge metagenomes. We have obtained from a sponge-bacterial association a complete pathway for the rare and potent antitumor agent psymberin from Psammocinia aff. bulbosa. The data support the symbiont hypothesis and provide insights into natural product evolution in previously inaccessible bacteria.

Somatic gain-of-function mutations of GNAQ and GNA11, which encode α subunits of heterotrimeric Gαq/11 proteins, occur in about 85% of cases of uveal melanoma (UM), the most common cancer of the adult eye. Molecular therapies to directly target these oncoproteins are lacking, and current treatment options rely on radiation, surgery, or inhibition of effector molecules downstream of these G proteins. A hallmark feature of oncogenic Gαq/11 proteins is their reduced intrinsic rate of hydrolysis of guanosine triphosphate (GTP), which results in their accumulation in the GTP-bound, active state. Here, we report that the cyclic depsipeptide FR900359 (FR) directly interacted with GTPase-deficient Gαq/11 proteins and preferentially inhibited mitogenic ERK signaling rather than canonical phospholipase Cβ (PLCβ) signaling driven by these oncogenes. Thereby, FR suppressed the proliferation of melanoma cells in culture and inhibited the growth of Gαq-driven UM mouse xenografts in vivo. In contrast, FR did not affect tumor growth when xenografts carried mutated B-RafV600E as the oncogenic driver. Because FR enabled suppression of malignant traits in cancer cells that are driven by activating mutations at codon 209 in Gαq/11 proteins, we envision that similar approaches could be taken to blunt the signaling of non-Gαq/11 G proteins.

In alfalfa nodules induced by Rhizobium meliloti strain L5-30 the compound L-3-0-methyl-scylloinosamine (3-0-MSI) is synthesized. This compound is also catabolized specifically by this strain. Its biological properties are therefore similar to the Agrobacterium opines. To answer the question whether opine-like compounds ("Rhizopines") play a role in a plant symbiotic interaction, we isolated the genes for the catabolism of 3-0-MSI (moc genes) and for the induction of its synthesis in the nodule [mos gene(s)]. moc and mos genes were shown to be closely linked and located on the Sym plasmid of L5-30, suggesting that they have co-evolved and may be important in symbiosis. These genes have been cloned into a broad host-range vector that can be mobilized into other R. meliloti strains where they are expressed. The location of the mos genes in the bacteria extends the opine concept, initially developed for a plant pathological interaction, to a symbiotic one.Plant-bacterial interactions are common in soil. The two best-studied examples are the pathological Agrobacterium-crowngall interaction and the symbiotic Rhizobiumlegume interaction. Agrobacterium and Rhizobium belong to the same family with Rhizobium meliloti regarded as being taxonomically the most similar to Agrobacterium (1). The pathological and symbiotic states induced by these two genera also have many features in common, including the ability to redirect plant morphogenesis and the presence of large plasmids involved in interaction with the plant (2, 3). Indeed, transfer of such plasmids from Rhizobium to Agrobacterium or vice versa results in expression of some of the plasmid-encoded symbiotic or pathogenic genes in the recipient host (see ref. 4).With the plant parasitic Agrobacterium-crowngall interaction the bacteria induce galls that act as factories to redirect plant metabolites to produce strain-specific, gall-specific compounds, called opines (5), which can be utilized by the inducing strain. The bacteria carry genes for the catabolism of these compounds and genes for their synthesis on the Ti plasmid. In this system the genes for opine synthesis are transferred and integrated, by way of the transferred DNA, into the plant genome (for a review, see ref. 6). The bacteria thus create an ecological niche, giving them a selective advantage over other bacteria. This phenomenon has been termed genetic colonization (7).In the Rhizobium-legume symbiosis plasmids also play an important role with many ofthe symbiotic genes (nod, nif, fix) being on a large plasmid (Sym plasmid) in a number of Rhizobium species, including R. meliloti, Rhizobium leguminosarum, and Rhizobium trifolii (8-11), but not in others, including Rhizobium loti (12).The development of symbiosis is a complex multistage process culminating in the formation of nodules in which bacteroids (differentiated bacteria) fix molecular nitrogen to ammonia. The plant obtains fixed nitrogen and supplies the bacteroids with photosynthetic products to fuel this process. It is generally accepted t...

We show that the promoter of the mos locus, which encodes genes required for the synthesis of a nodulespecific, opine-like compound, a rhizopine, in alfalfa nodules is regulated by the symbiotic nitrogen-fixation regulatory gene nifA. The 5'-regulatory region and amino-terminal end of the first open reading frame ofthe mos locus are highly homologous to the 5'-regulatory region and amino-terminal portion of the Rhizobium mefiloti niff gene. The coordinate regulation of mos and nifgenes suggests that the mos locus plays a symbiotic role. We propose that the rhizopine enhances the survival of the bacterial partner in the symbiosis.Recently, a nodule-specific, opine-like compound was isolated from alfalfa (Medicago sativa) nodules induced by Rhizobium meliloti strain L5-30 (1). This compound was described as opitne-like by analogy with Agrobacterium (2) since, of the strains tested, only the strain that induced its synthesis in the nodule could catabolize it as a growth substrate (1). The compound has been identified as L-3-0-methyl-scyllo-inosamine (3-0-MSI; ref.3). To distinguish nodule-specific, opine-like compounds from Agrobacterium opines, we have introduced the generic name rhizopine for such compounds (3).Previously, we isolated the mos genes, which are required for the synthesis of 3-0-MSI in the nodule, and the moc genes, which are necessary for 3-0-MSI catabolism by freeliving bacteria. We showed that these genes are closely linked and are on a large bacterial plasmid that carries a number of genes essential for symbiosis, including nodulation (nod and hsn) and nitrogen-fixation (nif and fix) genes (3). This suggested that the production and catabolism of 3-0-MSI has evolved as a functional unit and may be important in symbiosis. To investigate this notion further, we have studied the regulation of the mos genes to determine if they are symbiotically regulated.

The cyclic depsipeptide FR900359 (FR), isolated from the traditional Chinese medicine plant Ardisia crenata, is a potent Gq protein inhibitor and thus a valuable tool to study Gq-mediated signaling of G protein-coupled receptors. Two new FR analogues (3 and 4) were isolated from A. crenata together with the known analogues 1 and 2. The structures of compounds 3 and 4 were established by NMR spectroscopic data and MS-based molecular networking followed by in-depth LCMS analysis. The latter approach led to the annotation of further FR analogues 5-9. Comparative bioactivity tests of compounds 1-4 along with the parent molecule FR showed high-affinity binding to Gq proteins in the low nanomolar range (IC = 2.3-16.8 nM) for all analogues as well as equipotent inhibition of Gq signaling, which gives important SAR insights into this valuable natural product. Additionally, FR was detected from leaves of five other Ardisia species, among them the non-nodulated leaves of Ardisia lucida, implying a much broader distribution of FR than originally anticipated.

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Heterotrimeric G protein subunits Gαq and Gα11 are inhibited by two cyclic depsipeptides, FR900359 (FR) and YM-254890 (YM), both of which are being used widely to implicate Gq/11 proteins in the regulation of diverse biological processes. An emerging major research question therefore is whether the cellular effects of both inhibitors are on-target, that is, mediated via specific inhibition of Gq/11 proteins, or off-target, that is, the result of nonspecific interactions with other proteins. Here we introduce a versatile experimental strategy to discriminate between these possibilities. We developed a Gαq variant with preserved catalytic activity, but refractory to FR/YM inhibition. A minimum of two amino acid changes were required and sufficient to achieve complete inhibitor resistance. We characterized the novel mutant in HEK293 cells depleted by CRISPR–Cas9 of endogenous Gαq and Gα11 to ensure precise control over the Gα-dependent cellular signaling route. Using a battery of cellular outcomes with known and concealed Gq contribution, we found that FR/YM specifically inhibited cellular signals after Gαq introduction via transient transfection. Conversely, both inhibitors were inert across all assays in cells expressing the drug-resistant variant. These findings eliminate the possibility that inhibition of non-Gq proteins contributes to the cellular effects of the two depsipeptides. We conclude that combined application of FR or YM along with the drug-resistant Gαq variant is a powerful in vitro strategy to discern on-target Gq against off-target non-Gq action. Consequently, it should be of high value for uncovering Gq input to complex biological processes with high accuracy and the requisite specificity.