RESUMO
Bacterial secondary metabolites exhibit diverse remarkable bioactivities and are thus the subject of study for different applications. Recently, the individual effectiveness of tripyrrolic prodiginines and rhamnolipids against the plant-parasitic nematode Heterodera schachtii, which causes tremendous losses in crop plants, was described. Notably, rhamnolipid production in engineered Pseudomonas putida strains has already reached industrial implementation. However, the non-natural hydroxyl-decorated prodiginines, which are of particular interest in this study due to a previously described particularly good plant compatibility and low toxicity, are not as readily accessible. In the present study, a new effective hybrid synthetic route was established. This included the engineering of a novel P. putida strain to provide enhanced levels of a bipyrrole precursor and an optimization of mutasynthesis, i.e., the conversion of chemically synthesized and supplemented monopyrroles to tripyrrolic compounds. Subsequent semisynthesis provided the hydroxylated prodiginine. The prodiginines caused reduced infectiousness of H. schachtii for Arabidopsis thaliana plants resulting from impaired motility and stylet thrusting, providing the first insights on the mode of action in this context. Furthermore, the combined application with rhamnolipids was assessed for the first time and found to be more effective against nematode parasitism than the individual compounds. To obtain, for instance, 50% nematode control, it was sufficient to apply 7.8 µM hydroxylated prodiginine together with 0.7 µg/ml (~ 1.1 µM) di-rhamnolipids, which corresponded to ca. » of the individual EC50 values. In summary, a hybrid synthetic route toward a hydroxylated prodiginine was established and its effects and combinatorial activity with rhamnolipids on plant-parasitic nematode H. schachtii are presented, demonstrating potential application as antinematodal agents. Graphical Abstract.
RESUMO
High-level synthesis of complex enzymes like bacterial [NiFe] hydrogenases, in general, requires an expression system that allows concerted expression of a large number of genes. So far, it has not been possible to overproduce a hydrogenase in a stable and active form by using a customary expression system. Therefore we started to establish a new, T(7)-based expression system in the phototrophic bacterium Rhodobacter capsulatus. The beneficial properties of this bacterial host in combination with the unique capacity of T(7) RNA polymerase to synthesize long transcripts will allow the high-level synthesis and assembly of active hydrogenase as well as other complex enzymes in the near future.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Hidrogenase/genética , Rhodobacter sphaeroides/enzimologia , Transcrição Gênica , Proteínas Virais/genética , Clonagem Molecular , Hidrogenase/metabolismoRESUMO
Expression of nitrogen fixation genes in Rhodobacter capsulatus is repressed by ammonium at different regulatory levels including an NtrC-independent mechanism controlling NifA activity. In contrast to R. capsulatus NifA, heterologous NifA proteins of Klebsiella pneumoniae and Rhizobium meliloti, respectively, were not subjected to this posttranslational ammonium control in R. capsulatus. The characterization of ammonium-tolerant R. capsulatus NifA1 mutants indicated that the N-terminal domain of NifA was involved in posttranslational regulation. Analysis of a double mutant carrying amino acid substitutions in both the N-terminal domain and the C-terminal DNA-binding domain gave rise to the hypothesis that an interaction between these two domains might be involved in ammonium regulation of NifA activity. Western analysis demonstrated that both constitutively expressed wild-type and ammonium-tolerant NifA1 proteins exhibited high stability and accumulated to comparable levels in cells grown in the presence of ammonium excluding the possibility that proteolytic degradation was responsible for ammonium-dependent inactivation of NifA.