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In bacteria, many \"atypical\" response regulators (ARRs) lack the conserved residues important for phosphorylation by which typical response regulators switch their output response, suggesting the existence of alternative regulatory mechanisms. However, such mechanisms have not been established. JadR1, an OmpR-type ARR of Streptomyces venezuelae, appears to activate the transcription of jadomycin B (JdB) biosynthetic genes while repressing its own gene. JadR1 activities were inhibited in cells induced to produce JdB, which was found to bind directly to the N-terminal receiver domain of JadR1, causing JadR1 to dissociate from target promoters. The activity of a NarL-type ARR, RedZ, that regulates production of another antibiotic was likewise modulated by the end product (undecylprodigisines), implying that end-product-mediated control of antibiotic pathway-specific ARRs may be widespread. These results could prove relevant to knowledge-based improvements in yield of commercially important antibiotics.

In model organism Streptomyces coelicolor , γ-butyrolactones (GBLs) and antibiotics were recognized as signalling molecules playing fundamental roles in intra- and interspecies communications. To dissect the GBL and antibiotic signalling networks systematically, the in vivo targets of their respective receptors ScbR and ScbR2 were identified on a genome scale by ChIP-seq. These identified targets encompass many that are known to play important roles in diverse cellular processes (e.g. gap1 , pyk2 , afsK , nagE2 , cdaR , cprA , cprB , absA1 , actII-orf4 , redZ , atrA , rpsL and sigR ) and they formed regulatory cascades, sub-networks and feedforward loops to elaborately control key metabolite processes, including primary and secondary metabolism, morphological differentiation and stress response. Moreover, interplay among ScbR, ScbR2 and other regulators revealed intricate cross talks between signalling pathways triggered by GBLs, antibiotics, nutrient availability and stress. Our work provides a global view on the specific responses that could be triggered by GBL and antibiotic signals in S. coelicolor , among which the main echo was the change of production profile of endogenous antibiotics and antibiotic signals manifested a role to enhance bacterial stress tolerance as well, shedding new light on GBL and antibiotic signalling networks widespread among streptomycetes.

Background Oxytetracycline (OTC) is a broad-spectrum antibiotic commercially produced by Streptomyces rimosus . Despite its importance, little is known about the regulation of OTC biosynthesis, which hampered any effort to improve OTC production via engineering regulatory genes. Results A gene encoding a Streptomyces antibiotic regulatory protein (SARP) was discovered immediately adjacent to the otrB gene of oxy cluster in S. rimosus and designated otcR . Deletion and complementation of otcR abolished or restored OTC production, respectively, indicating that otcR encodes an essential activator of OTC biosynthesis. Then, the predicted consensus SARP-binding sequences were extracted from the promoter regions of oxy cluster. Transcriptional analysis in a heterologous GFP reporter system demonstrated that OtcR directly activated the transcription of five oxy promoters in E. coli , further mutational analysis of a SARP-binding sequence of oxyI promoter proved that OtcR directly interacted with the consensus repeats. Therefore, otcR was chosen as an engineering target, OTC production was significantly increased by overexpression of otcR as tandem copies each under the control of strong SF14 promoter. Conclusions A SARP activator, OtcR, was identified in oxy cluster of S. rimosus ; it was shown to directly activate five promoters from oxy cluster. Overexpression of otcR at an appropriate level dramatically increased OTC production by 6.49 times compared to the parental strain, thus demonstrating the great potential of manipulating OtcR to improve the yield of OTC production.

The naturally occurring diazobenzofluorenes, kinamycins, fluostatins and lomaiviticins, possess highly oxygenated A-rings, via which the last forms a dimeric pharmacophore. However, neither the A-ring transformation nor the dimerization mechanisms have been explored thus far. Here we propose a unified biosynthetic logic for the three types of antibiotics and verify one key reaction via detailed genetic and enzymatic experiments. Alp1U and Lom6 from the kinamycin and lomaiviticin biosynthesis, respectively, are shown to catalyse epoxy hydrolysis on a substrate that is obtained by chemical deacetylation of a kinamycin-pathway-derived intermediate. Thus, our study provides the first evidence for the existence of an epoxy intermediate in lomaiviticin biosynthesis. Furthermore, our results suggest that the dimerization in the lomaiviticin biosynthesis proceeds after dehydration of a product generated by Lom6.

The jadomycins are a unique family of angucycline-derived antibiotics with interesting cytotoxic activities. In this work, six new jadomycin derivatives were produced in vivo by providing non-natural amino acids in fermentation media. They were further purified and identified by MS and NMR analyses. The cytotoxicities of these derivatives were evaluated against tumor cell lines MCF-7 and HCT116, as well as the normal human microvascular epithelial cells. The derivatives with alkyl side chains showed similar levels of cytotoxicity as jadomycin B and other known derivatives with nonpolar side chains, with IC 50 ranging from 1.3 to 10 μ M ; but the activities are not selective as these compounds also showed similar levels of cytotoxicity toward the normal human microvascular epithelial cells in the same concentration range. For the first time, derivatives with amino side chains (jadomycin Orn and K) were prepared and evaluated. Significantly, jadomycin Orn showed differential activity against normal and tumor cell lines. This result points to a new direction to modify jadomycin structure. The insights on the structure–activity relationship of jadomycins will guide further efforts to generate new and improved jadomycin derivatives against tumor cells.

The high mortality rate of immunocompromised patients with fungal infections and the limited availability of highly efficacious and safe agents demand the development of new antifungal therapeutics. To rapidly discover such agents, we developed a high-throughput synergy screening (HTSS) strategy for novel microbial natural products. Specifically, a microbial natural product library was screened for hits that synergize the effect of a low dosage of ketoconazole (KTC) that alone shows little detectable fungicidal activity. Through screening of [almost equal to]20,000 microbial extracts, 12 hits were identified with broad-spectrum antifungal activity. Seven of them showed little cytotoxicity against human hepatoma cells. Fractionation of the active extracts revealed beauvericin (BEA) as the most potent component, because it dramatically synergized KTC activity against diverse fungal pathogens by a checkerboard assay. Significantly, in our immunocompromised mouse model, combinations of BEA (0.5 mg/kg) and KTC (0.5 mg/kg) prolonged survival of the host infected with Candida parapsilosis and reduced fungal colony counts in animal organs including kidneys, lungs, and brains. Such an effect was not achieved even with the high dose of 50 mg/kg KTC. These data support synergism between BEA and KTC and thereby a prospective strategy for antifungal therapy.

Increasing the self-resistance levels of Streptomyces is an effective strategy to improve the production of antibiotics.To increase the oxytetracycline（OTC） production in Streptomyces rimosus,we investigated the cooperative effect of three co-overexpressing OTC resistance genes：one gene encodes a ribosomal protection protein（otrA） and the other two express efflux proteins（otrB and otrC）.Results indicated that combinational overexpression of otrA,otrB,and otrC（MKABC） exerted a synergetic effect.OTC production increased by 179%in the recombinant strain compared with that of the wild-type strain M4018.The resistance level to OTC was increased by approximately two-fold relative to the parental strain,thereby indicating that applying the cooperative effect of self-resistance genes is useful to improve OTC production.Furthermore,the previously identified cluster-situated activator OtcR was overexpressed in MKABC in constructing the recombinant strain MKRABC;such strain can produce OTC of approximately7.49 g L~（（-1））,which represents an increase of 19%in comparison with that of the OtcR-overexpressing strain alone.Our work showed that the cooperative overexpression of self-resistance genes is a promising strategy to enhance the antibiotics production in Streptomyces.

The glycerol utilization （gyl） operon is involved in clavulanic acid （CA） production by Streptomyces clavuligerus, and possibly supplies the glyceraldehyde-3-phosphate （G3P） precursor for CA biosynthesis. The gyl operon is regulated by GylR and is induced by glycerol. To enhance CA production in S. clavuligerus, an extra copy of ccaR expressed from Pgyl （the gyl promoter） was integrated into the chromosome of S. clavuligerus NRRL 3585. This construct coordinated the transcription of CA biosynthetic pathway genes with expression of the gyl operon. In the transformants carrying the Pgyl-controlled regulatory gene ccaR, CA production was enhanced 3.19-fold in glycerol-enriched batch cultures, relative to the control strain carrying an extra copy of ccaR controlled by its own promoter （PccaR）. Consistent with enhanced CA production, the transcription levels of ccaR, ceas2 and claR were significantly up-regulated in the transformants containing Pgyl-controlled ccaR.

The angucycline antibiotic jadomycin B (JdB) produced by Streptomyces venezuelae has been found here to induce complex survival responses in Streptomyces coelicolor at subinhibitory concentration. The receptor for JdB was identified as a “pseudo” gamma-butyrolactone receptor, ScbR2, which was shown to bind two previously unidentified target promoters, those of redD (redD p) and adpA (adpA p), thus directly regulating undecylprodigiosin (Red) production and morphological differentiation, respectively. Because AdpA also directly regulates the expression of redD , ScbR2, AdpA, and RedD together form a feed-forward loop controlling both differentiation and Red production phenotypes. Different signal strengths (i.e., JdB concentrations) were shown to induce the two different phenotypes by modulating the relative transcription levels of adpA vs. redD . The induction of morphological differentiation and endogenous antibiotic production by exogenous antibiotic exemplifies an important survival strategy more sophisticated than the induction of antibiotic resistance.

We developed a colorimetric assay to quantify clavulanic acid （CA） in culture broth of Streptomyces clavuligerus, to facilitate screening of a large number of S. clavuligerus mutants. The assay is based on a β-1actamase-catalyzed reaction, in which the yellow substrate nitrocefin （λmax=390 nm） is converted to a red product （λmax=486 nm）. Since CA can irreversibly inhibit β-1actamase activity, the level of CA in a sample can be measured as a function of the A390]A486 ratio in the assay mixture. The sensitivity and detection window of the assay were determined to be 50 μg L -1 and 50 μg L to 10 mg L-1, respectively. The reliability of the assay was confirmed by comparing assay results with those obtained by HPLC. The assay was used to screen a pool of 65 S. clavuligerus mutants and was reliable for identifying CA over-producing mutants. Therefore, the assay saves time and labor in large-scale mutant screening and evaluation tasks. The detection window and the reliability of this assay are markedly better than those of previously reported CA assays. This assay method is suitable for high throughput screening of microbial samples and allows direct visual observation of CA levels on agar plates.