Background Elaboration of the epigenetic regulation of chromatin is a long-standing aim in molecular and cellular biology. Hence, there is a great demand for the development of in vitro methods to reconstitute chromatin that can be used directly for biochemical assays. The widely used wheat germ cell-free protein expression method provides broad applications to investigate the function and structure of eukaryotic proteins. Such advantages, including high translation efficiency, flexibility, and possible automatization, are beneficial for achieving native-like chromatin substrates for in vitro studies. Results We describe a novel, single-step in vitro chromatin assembly method by using the wheat germ cell-free protein synthesis. We demonstrated that both Drosophila and human chromatins can be reconstituted in the course of the in vitro translation of core histones by the addition of chromatin assembly factors, circular plasmid, and topoisomerase I in an ATP-dependent manner. Drosophila chromatin assembly was performed in 4 h at 26 °C, in the presence of premixed mRNAs encoding the core histones, dAcf1/dISWI chromatin remodeling complex, and nucleosome assembly protein, dNAP1. Similarly, the human chromatin was assembled by co-expressing the human core histones with Drosophila chromatin remodeling factor, dISWI, and chromatin chaperone, dNLP, for 6 h at 26 °C. The presence of reconstituted chromatin was monitored by DNA supercoiling assay, also the regular spacing of nucleosomes was assessed by Micrococcal nuclease assay. Furthermore, Drosophila linker histone H1-containing chromatin was reconstituted, affirming that the in vitro assembled chromatin is suitable for downstream applications. Conclusions The method described in this study allows the assembly of Drosophila and human chromatins, possibly in native-like form, by using a wheat germ cell-free protein expression. Although both chromatins were reconstituted successfully, there were unexpected differences with respect to the required ratio of histone-coding mRNAs and the reaction time. Overall, our new in vitro chromatin reconstitution method will aid to characterize the unrevealed structure, function, and regulation of chromatin dynamics.

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The cellulolytic insect symbiont bacterium, Streptomyces sp. SirexAA-E (SirexAA-E) secretes a suite of Carbohydrate Active enZymes (CAZymes), which are involved in the degradation of various polysaccharides in the plant cell wall, in response to the available carbon sources. Here, we examined a poorly understood response of this bacterium to mannan, one of the major plant cell wall components. SirexAA-E grew well on mannose, carboxymethyl cellulose (CMC), and locust bean gum (LBG) as sole carbon sources in the culture medium. The secreted proteins from each culture supernatant were tested for their polysaccharide-degrading ability, and the composition of secreted CAZymes in each sample was determined by LC-MS/MS. The results indicated that mannose, LBG, and CMC induced the secretion of mannan and cellulose-degrading enzymes. Interestingly, two α-1,2-mannosidases were abundantly secreted during growth on mannose and LBG. By genomic analysis, we found a unique 12 bp palindromic sequence motif at 4 locations in the SirexAA-E genome, two of which were found upstream of the above-mentioned α-1,2-mannosidase genes, along with a newly identified mannose and mannobiose-responsive transcriptional regulator, SsManR. Furthermore, the previously reported cellobiose-responsive repressor, SsCebR, was determined to also use mannobiose as an effector ligand. To test whether mannobiose induces the sets of genes under the control of the two regulators, SirexAA-E was grown on mannobiose, and the secretome composition was analyzed. As hypothesized, the composition of the mannobiose secretome combined sets of CAZymes found in both LBG and CMC secretomes, and so are likely under the regulation of both SsManR and SsCebR. Importance Streptomyces sp. SirexAA-E, a microbial symbiont of biomass harvesting insects, secretes a suite of polysaccharide-degrading enzymes dependent on the available carbon sources. However, the response of this bacterium to mannan has not been documented. In this study, we investigated the response of this bacterium to mannose, mannobiose, and galactomannan (LBG). By combining biochemical, proteomic, and genomic approaches, we discovered a novel mannose and mannobiose responsive transcriptional regulator, SsManR, which selectively regulates three α-1,2-mannosidase-coding genes. We also demonstrated that the previously described cellobiose responsive regulator, SsCebR, could use mannobiose as an effector ligand. Overall, our findings suggest that the Streptomyces sp. SirexAA-E responds to mannose and mannooligosaccharides through two different transcriptional repressors that regulate the secretion of the plant cell wall-degrading enzymes to extract carbon sources in the host environment.

The nucleosome, a basic unit of chromatin found in all eukaryotes, is thought to be assembled through the orchestrated activity of several histone chaperones and chromatin assembly factors in a stepwise manner, proceeding from tetrasome assembly, to H2A/H2B deposition, and finally to formation of the mature nucleosome. In this study, we demonstrate chaperone‐mediated assembly of both tetrasomes and nucleosomes on the well‐defined Widom 601 positioning sequence using a co‐expression/reconstitution wheat germ cell‐free system. The purified tetrasomes and nucleosomes were positioned around the center of a given sequence. The heights and diameters were measured by atomic force microscopy. Together with the reported unmodified native histones produced by the wheat germ cell‐free platform, our method is expected to be useful for downstream applications in the field of chromatin research.