Sharief Barends

Feast or famine: the global regulator DasR links nutrient stress to antibiotic production by Streptomyces

Members of the soil‐dwelling prokaryotic genus Streptomyces produce many secondary metabolites, including antibiotics and anti‐tumour agents. Their formation is coupled with the onset of development, which is triggered by the nutrient status of the habitat. We propose the first complete signalling cascade from nutrient sensing to development and antibiotic biosynthesis. We show that a high concentration of N‐acetylglucosamine—perhaps mimicking the accumulation of N‐acetylglucosamine after autolytic degradation of the vegetative mycelium—is a major checkpoint for the onset of secondary metabolism. The response is transmitted to antibiotic pathway‐specific activators through the pleiotropic transcriptional repressor DasR, the regulon of which also includes all N‐acetylglucosamine‐related catabolic genes. The results allowed us to devise a new strategy for activating pathways for secondary metabolite biosynthesis. Such ‘cryptic’ pathways are abundant in actinomycete genomes, thereby offering new prospects in the fight against multiple drug‐resistant pathogens and cancers.

The Square-Planar Cytotoxic [CuII(pyrimol)Cl] Complex Acts as an Efficient DNA Cleaver without Reductant

Chemical nucleases based on the transition-metal ions cleave DNA hydrolytically and/or oxidatively, with or without added reductant. We report here the novel DNA cleavage properties of the highly water-soluble, square-planar [Cu(Hpyrimol)Cl] complex, together with the results of cytotoxicities toward selected cancer cell lines. The copper complex cleaves PhiX174 supercoiled DNA efficiently without any reductant and shows high cytotoxicities toward L1210 murine leukemia and A2780 human ovarian carcinoma cancer cell lines that are sensitive and resistant to cisplatin. The IC50 values obtained for the copper complex in the sensitive cell lines are in the range of cisplatin, and for the cisplatin-resistant leukemia cell line, this value is even better.

Structure, Cytotoxicity, and DNA-Cleavage Properties of the Complex [CuII(pbt)Br2]

The reactions of the ligand 2-(2-pyridyl)benzthiazole (pbt) with CuBr 2 and ZnCl 2 in acetonitrile produce the complexes [Cu(pbt)Br 2] ( 1) and [Zn(pbt)Cl 2] ( 3), respectively. When complex 1 is dissolved in DMF, complex 2 is obtained as light-green crystals. The reaction of pbt with CuBr 2 in DMF also yields the complex [Cu(pbt)Br 2(dmf)] ( 2) (dmf = dimethylformamide). Complexes 1- 3 were characterized by X-ray crystallography. Complexes 1 and 3 have distorted tetrahedral coordination environments, and complex 2 is constituted of two slightly different copper centers, both exhibiting distorted trigonal bipyramidal geometries. Complexes 1 and 2 cleave phiX174 phage DNA, both in the presence and the absence of reductant. The free ligand pbt does not show any DNA-cleaving abilities. The poor solubility of complex 3 makes it not applicable for biological tests. The occurrence of DNA breaks in the presence of various radical scavengers suggests that no diffusible radicals are involved in the DNA cleavage by complex 1, as none of the scavengers inhibit the cleavage reaction. The DNA-cleavage products are not religated with the enzyme T4 DNA ligase, which is an additional proof that the cleavage is nonhydrolytic. Most probably the cleaving reaction involves reactive oxygen species, which could not be trapped, leading to an oxidative mechanism. An easy oxidation of Cu (II)(pbt)Br 2 to Cu (III) in DMF and the reduction of the same to Cu (I), under similar electrochemical conditions may lead to the in situ activation of molecular oxygen, resulting in the formation of metal solvated nondiffusible radicals able to prompt the oxidative cleavage of DNA. Complex 1 and the pure ligand exhibit remarkable cytotoxic effects against the cancer cell lines L1210 and A2780 and also against the corresponding cisplatin-resistant mutants of these cell lines.

tRNA-Like Structure Regulates Translation of Brome Mosaic Virus RNA

ABSTRACT For various groups of plant viruses, the genomic RNAs end with a tRNA-like structure (TLS) instead of the 3′ poly(A) tail of common mRNAs. The actual function of these TLSs has long been enigmatic. Recently, however, it became clear that for turnip yellow mosaic virus, a tymovirus, the valylated TLSTYMV of the single genomic RNA functions as a bait for host ribosomes and directs them to the internal initiation site of translation (with N-terminal valine) of the second open reading frame for the polyprotein. This discovery prompted us to investigate whether the much larger TLSs of a different genus of viruses have a comparable function in translation. Brome mosaic virus (BMV), a bromovirus, has a tripartite RNA genome with a subgenomic RNA4 for coat protein expression. All four RNAs carry a highly conserved and bulky 3′ TLSBMV (about 200 nucleotides) with determinants for tyrosylation. We discovered TLSBMV-catalyzed self-tyrosylation of the tyrosyl-tRNA synthetase but could not clearly detect tyrosine incorporation into any virus-encoded protein. We established that BMV proteins do not need TLSBMV tyrosylation for their initiation. However, disruption of the TLSs strongly reduced the translation of genomic RNA1, RNA2, and less strongly, RNA3, whereas coat protein expression from RNA4 remained unaffected. This aberrant translation could be partially restored by providing the TLSBMV in trans. Intriguingly, a subdomain of the TLSBMV could even almost fully restore translation to the original pattern. We discuss here a model with a central and dominant role for the TLSBMV during the BMV infection cycle.

Pip, a Novel Activator of Phenazine Biosynthesis in Pseudomonas chlororaphis PCL1391

Secondary metabolites are important factors for interactions between bacteria and other organisms. Pseudomonas chlororaphis PCL1391 produces the antifungal secondary metabolite phenazine-1-carboxamide (PCN) that inhibits growth of Fusarium oxysporum f. sp. radius lycopersici the causative agent of tomato foot and root rot. Our previous work unraveled a cascade of genes regulating the PCN biosynthesis operon, phzABCDEFGH. Via a genetic screen, we identify in this study a novel TetR/AcrR regulator, named Pip (phenazine inducing protein), which is essential for PCN biosynthesis. A combination of a phenotypical characterization of a pip mutant, in trans complementation assays of various mutant strains, and electrophoretic mobility shift assays identified Pip as the fifth DNA-binding protein so far involved in regulation of PCN biosynthesis. In this regulatory pathway, Pip is positioned downstream of PsrA (Pseudomonas sigma factor regulator) and the stationary-phase sigma factor RpoS, while it is upstream of the quorum-sensing system PhzI/PhzR. These findings provide further evidence that the path leading to the expression of secondary metabolism gene clusters in Pseudomonas species is highly complex.

Functional evidence for D- and T-loop interactions in tmRNA

During bacterial protein synthesis, stalled ribosomes can be rescued by tmRNA, a molecule with both tRNA and mRNA features. The tRNA region of tmRNA has sequence similarity with tRNAAla and also has a clover‐leaf structure folded similarly as in canonical tRNAs. Here we propose the L‐shape of tmRNA to be stabilized by two tertiary interactions between its D‐ and T‐loop on the basis of phylogenetic and experimental evidence. Mutational analysis clearly demonstrates a tertiary interaction between G13 and U342. Strikingly, this in evolution conserved interaction is not primarily important for tmRNA alanylation and for binding to elongation factor Tu, but especially for a proper functioning of SmpB.

The tmRNA‐tagging mechanism and the control of gene expression: a review

The tmRNA‐mediated trans‐translation system is a unique quality control system in eubacteria that combines translational surveillance with the rescue of stalled ribosomes. During trans‐translation, the chimeric tmRNA molecule—which acts as both tRNA and mRNA—is delivered to the ribosomal A site by a ribonucleoprotein complex of SmpB and EF‐Tu–GTP, allowing the stalled ribosome to switch template and resume translation on a small coding sequence inside the tmRNA molecule. As a result, the aberrant protein becomes tagged by a sequence that is a target for proteolytic degradation. Thus, the system elegantly combines ribosome recycling with a clean‐up function when triggered by truncated transcripts or rare codons. In addition, recent observations point to a specific regulation of the translation of a small number of genes by tmRNA‐mediated inhibition or stimulation. In this review, we discuss the most prominent biochemical and structural aspects of trans‐translation and then focus on the specific role of tmRNA in stress management and cell‐cycle control of morphologically complex bacteria. WIREs RNA 2011 2 233–246 DOI: 10.1002/wrna.48

Transfer–messenger RNA controls the translation of cell‐cycle and stress proteins in Streptomyces

The transfer–messenger RNA (tmRNA)‐mediated trans‐translation mechanism is highly conserved in bacteria and functions primarily as a system for the rescue of stalled ribosomes and the removal of aberrantly produced proteins. Here, we show that in the antibiotic‐producing soil bacterium Streptomyces coelicolor, trans‐translation has a specialized role in stress management. Analysis of proteins that were carboxy‐terminally His8‐tagged by a recombinant tmRNA identified only 10 targets, including the stress proteins: DnaK heat‐shock protein 70, thiostrepton‐induced protein A, universal stress protein A, elongation factor Tu3, and the cell‐cycle control proteins DasR, SsgA, SsgF and SsgR. Although tmRNA‐tagged proteins are degraded swiftly, the translation of dnaK and dasR messenger RNAs (mRNAs) depends fully on tmRNA, whereas transcription is unaffected. The data unveil a surprisingly dedicated functionality for tmRNA, promoting the translation of the same mRNA it targets, at the expense of sacrificing the first nascent protein. In streptomycetes, tmRNA has evolved into a dedicated task force that ensures the instantaneous response to the exposure to stress.