Summary In Streptomyces, GlnR is an activator protein that activates nitrogen‐assimilation genes under nitrogen‐limiting conditions. However, less is known regarding the regulation of these genes under nitrogen‐rich conditions. We determined that the developmental regulator MtrA represses nitrogen‐assimilation genes in nitrogen‐rich media and that it competes with GlnR for binding to GlnR boxes. The GlnR boxes upstream of multiple nitrogen genes, such as amtB, were confirmed as MtrA binding sites in vitro by electrophoretic mobility shift assays and in vivo by ChIP‐qPCR analysis. Transcriptional analysis indicated that, on nutrient‐rich medium, MtrA profoundly repressed expression of nitrogen‐associated genes, indicating opposing roles for MtrA and GlnR in the control of nitrogen metabolism. Using in vitro and in vivo analysis, we also showed that glnR is itself a direct target of MtrA and that MtrA represses glnR transcription. We further demonstrated functional conservation of MtrA homologues in the recognition of GlnR boxes upstream of nitrogen genes from different actinobacterial species. As mtrA and glnR are widespread among actinomycetes, this mechanism of potential competitive control over nitrogen metabolism genes may be common in this group, adding a major new layer of complexity to the known regulatory network for nitrogen metabolism in Streptomyces and related species.

In order to reveal the mechanisms of the extreme radioresistance and DNA repair in Deinococcus radiodurans, we examined proteome changes in a wild-type strain following gamma-irradiation using two-dimensional polyacrylamide gel electrophoresis and Silver-staining. The expression levels of 26 protein spots showed significant changes under radiation stress. Of these spots, 21 were identified with peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight mass spectrometry after tryptic in-gel digestion. These proteins exhibited various cellular functions, including (i) translation; (ii) transcription; (iii) signal transduction; (iv) post-translational modification, protein turnover, chaperones; (v) carbohydrate transport and metabolism; (vi) energy production and conversion; (vii) nucleotide transport and metabolism; (viii) inorganic ion transport and metabolism; (ix) DNA replication, recombination and repair; and (x) yet unknown. Most of the proteins have not previously been reported to be relevant to radioresistance.

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Hjm and Hel308a are novel, RecQ-like DNA helicases recently identified in the euryarchaeotes Pyrococcus furiosus and Methanothermobacter thermautotrophicus, respectively. In this study, an Hjm/Hel308 homologue (designated StoHjm) from Sulfolobus tokodaii, a hyperthermophilic archaeon belonging to the Crenarchaeota subdomain of archaea, was cloned, purified, and characterized. Unlike Hjm and Hel308a, which unwind DNA in a 3-to-5 direction, StoHjm unwound DNA in both 3-to-5 and 5-to-3 directions. Remarkably, StoHjm exhibited structure-specific single-stranded-DNA-annealing and fork regression activities in vitro. In addition, gel filtration, affinity pulldown, and yeast two-hybrid analyses revealed that StoHjm physically interacted with StoHjc, the Holliday junction-specific endonuclease from S. tokodaii. This interaction may have functional significance, because the unwinding activity of StoHjm was inhibited by StoHjc in vitro. These results may suggest that the Hjm/Hel308 family helicases, in association with Hjc endonucleases, are involved in processing of stalled replication forks.

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BackgroundThe CRISPR/dCas9 system is a powerful tool to activate the transcription of target genes in eukaryotic or prokaryotic cells, but lacks assays in complex conditions, such as the biosynthesis of secondary metabolites.ResultsIn this study, to improve the transcription of the heterologously expressed biosynthetic genes for the production of epothilones, we established the CRISPR/dCas9-mediated activation technique in Myxococcus xanthus and analyzed some key factors involving in the CRISPR/dCas9 activation. We firstly optimized the cas9 codon to fit the M. xanthus cells, mutated the gene to inactivate the nuclease activity, and constructed the dCas9-activator system in an epothilone producer. We compared the improvement efficiency of different sgRNAs on the production of epothilones and the expression of the biosynthetic genes. We also compared the improvement effects of different activator proteins, the ω and α subunits of RNA polymerase, and the sigma factors σ54 and CarQ. By using a copper-inducible promoter, we determined that higher expressions of dCas9-activator improved the activation effects.ConclusionsOur results showed that the CRISPR/dCas-mediated transcription activation is a simple and broadly applicable technique to improve the transcriptional efficiency for the production of secondary metabolites in microorganisms. This is the first time to construct the CRISPR/dCas9 activation system in myxobacteria and the first time to assay the CRISPR/dCas9 activations for the biosynthesis of microbial secondary metabolites.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0867-1) contains supplementary material, which is available to authorized users.

The protein DRA0074 is suggested to be another LexA in Deinococcus radiodurans, having similar motifs and RecA-mediated cleavage activity to D. radiodurans LexA (dra0344). However, its function has not been studied. We disrupted the gene dra0074 and measured its effect on RecA induction using fusion translation, immunoblot, and proteomic analysis. Results showed that the product of gene dra0074 is not involved in RecA induction, but is a regulator of other metabolisms in D. radiodurans.