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.

SummaryMembers of the soil-dwelling, sporulating prokaryotic genus Streptomyces are indispensable for the recycling of the most abundant polysaccharides on earth (cellulose and chitin), and produce a wide range of antibiotics and industrial enzymes. How do these organisms sense the nutritional state of the environment, and what controls the signal for the switch to antibiotic production and morphological development? Here we show that high extracellular concentrations of N-acetylglucosamine, the monomer of chitin, prevent Streptomyces coelicolor progressing beyond the vegetative state, and that this effect is absent in a mutant defective of N-acetylglucosamine transport. We provide evidence that the signal is transmitted through the GntR-family regulator DasR, which controls the N-acetylglucosamine regulon, including the pts genes ptsH, ptsI and crr needed for uptake of N-acetylglucosamine. Deletion of dasR or the pts genes resulted in a bald phenotype. Binding of DasR to its target genes is abolished by glucosamine 6-phosphate, a central molecule in N-acetylglucosamine metabolism. Extracellular complementation experiments with many bld mutants showed that the dasR mutant is arrested at an early stage of the developmental programme, and does not fit in the previously described bld signalling cascade. Thus, for the first time we are able to directly link carbon (and nitrogen) metabolism to development, highlighting a novel type of metabolic regulator, which senses the nutritional state of the habitat, maintaining vegetative growth until changing circumstances trigger the switch to sporulation. Our work, and the model it suggests, provide new leads towards understanding how microorganisms time developmental commitment.

SummaryHPr(Ser) kinase is the sensor in a multicomponent phosphorelay system that controls catabolite repression, sugar transport and carbon metabolism in Grampositive bacteria. Unlike most other protein kinases, it recognizes the tertiary structure in its target protein, HPr, a phosphocarrier protein of the bacterial phosphotransferase system and a transcriptional cofactor controlling the phenomenon of catabolite repression. We have identified the gene (ptsK ) encoding this serine/threonine protein kinase and characterized the purified protein product. Orthologues of PtsK have been identified only in bacteria. These proteins constitute a novel family unrelated to other previously characterized protein phosphor ylating enzymes. The Bacillus subtilis kinase is shown to be allosterically activated by metabolites such as fructose 1,6-bisphosphate and inhibited by inorganic phosphate. In contrast to wild-type B. subtilis, the ptsK mutant is insensitive to transcriptional regulation by catabolite repression. The reported results advance our understanding of phosphor ylation-dependent carbon control mechanisms in Gram-positive bacteria.

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Alterations of the proto-oncogene MLL (mixed lineage leukemia) are characteristic for a high proportion of acute leukemias, especially those occurring in infants. The activation of MLL is achieved either by an internal tandem duplication of 5' MLL exons or by chromosomal translocations that create chimeric proteins with the N-terminus of MLL fused to a variety of different partner proteins. A domain of MLL with significant homology to the eukaryotic DNA methyltransferases (MT domain) has been found to be essential for the transforming potential of the oncogenic MLL derivatives. Here we demonstrate that this domain specifically recognizes DNA with unmethylated CpG sequences. In gel mobility shifts, the presence of CpG was sufficient for binding of recombinant GST-MT protein to DNA. The introduction of 5-methylCpG on one or both DNA strands precluded an efficient interaction. In surface plasmon resonance a KD of approximately 3.3 x 10(-8) M was determined for the GST-MT/DNA complex formation. Site selection experiments and DNase I footprinting confirmed CpG as the target of the MT domain. Finally, this interaction was corroborated in vivo in reporter assays utilizing the DNA-binding properties of the MT domain in a hybrid MT-VP16 transactivator construct.