The discovery of Streptomyces-produced streptomycin founded the age of tuberculosis therapy. Despite the subsequent development of a curative regimen for this disease, tuberculosis remains a worldwide problem, and the emergence of multidrug-resistant Mycobacterium tuberculosis has prioritized the need for new drugs. Here we show that new optimized derivatives from Streptomyces-derived griselimycin are highly active against M. tuberculosis, both in vitro and in vivo, by inhibiting the DNA polymerase sliding clamp DnaN. We discovered that resistance to griselimycins, occurring at very low frequency, is associated with amplification of a chromosomal segment containing dnaN, as well as the ori site. Our results demonstrate that griselimycins have high translational potential for tuberculosis treatment, validate DnaN as an antimicrobial target, and capture the process of antibiotic pressure-induced gene amplification.

The broad-host-range plasmid RP4 encodes a highly efficient partitioning system (par) that was previously mapped within the 6.2-kb PstI C fragment. The essential functions were assigned to a region of 2.2 kb between fiwA and IS21 (IS8). On the basis of the nucleotide sequence data of the entire par locus and of in vitro and in vivo expression studies, three distinct loci encoding polypeptides of 9, 18, and 24 kDa were identified. Evidence for the expression of another polypeptide was found. A putative divergent promoter was localized in an intergenic region and is suggested to be responsible for transcription of these genes. It was found that the RP4 par region includes a function resolving plasmid dimers. The 24-kDa polypeptide is considered to function as a resolvase, since its predicted amino acid sequence shows homology to sequences of resolvases of the Tn3 family. Furthermore, palindromes present in the intergenic region containing the divergent promoter resemble repeat structures specific for res sites of Tn3-related transposons. However, it was found that dimer resolution itself was not sufficient for stabilization; additional functions, including the other two polypeptides, seemed to play an important role. These results suggested that RP4 contains a complex stabilization system involving resolution of plasmid dimers during cell division, thus ensuring the delivery of at least one copy to each daughter cell.

Wild-type p53 plays a crucial role in the prevention of cancer. Since dysfunction of p53 can be caused by increased levels of the protein MDM2, small molecules which antagonize the interaction between these two proteins have potential in cancer therapy. The discovery and structure determination of a fungal metabolite, chlorofusin, which antagonizes the p53/MDM2 interaction are reported.

To examine as randomly as possible the role of the ␤-ketoacyl and acyl carrier protein (ACP) components of bacterial type II polyketide synthases (PKSs), homologs of the chain-length-factor (CLF) genes were cloned from the environmental community of microorganisms. With PCR primers derived from conserved regions of known ketosynthase (KS ␣ ) and ACP genes specifying the formation of 16-to 24-carbon polyketides, two CLF (KS ␤ ) genes were cloned from unclassified streptomycetes isolated from the soil, and two were cloned from soil DNA without the prior isolation of the parent microorganism. The sequence and deduced product of each gene were distinct from those of known KS ␤ genes and, by phylogenetic analysis, belonged to antibiotic-producing PKS gene clusters. Hybrid PKS gene cassettes were constructed with each novel KS ␤ gene substituted for the actI-ORF2 or tcmL KS ␤ subunit genes, along with the respective actI-ORF1 or tcmK KS ␣ , tcmM ACP, and tcmN cyclase genes, and were found to produce an octaketide or decaketide product characteristic of the ones known to be made by the heterologous KS ␣ gene partner. Since substantially less than 1% of the microorganisms present in soil are thought to be cultivatable by standard methods, this work demonstrates a potential way to gain access to a more extensive range of microbial molecular diversity and to biosynthetic pathways whose products can be tested for biological applications.The polyketide metabolites are a large group of structurally complex and diverse natural products, many of which are clinically valuable antibiotics or chemotherapeutic agents or have other useful pharmacological activities (48). Polyketides are synthesized by polyketide synthases (PKSs) which, like the related fatty acid synthases, are multifunctional enzyme assemblies that catalyze repeated decarboxylative condensations between enzyme-bound acylthioesters (52). The structural diversity of polyketides is a result of the different numbers and types of acyl units involved, coupled with various possibilities for enzyme-mediated reduction, dehydration, cyclization, and aromatization of the initial ␤-ketoacyl condensation products (52). Manipulation of the sequence or specificity of the PKS enzymes can lead to the production of novel secondary metabolites (28,29,33). The key to further exploitation of this approach to molecular diversity lies in an increased understanding of the genetics and biochemistry of the various PKS systems.Recent progress in the cloning and sequencing of microbial PKS genes has led to the identification of at least two architecturally different types of PKSs (28, 33). Modular type I PKSs, represented by erythromycin A (11, 17), avermectin (41), rapamycin (58), and soraphen (57), are discrete multifunctional enzymes sporting numerous unique active site domains. The domains are grouped into distinct modules that in turn control the sequential addition of acylthioester units to the growing polyketide chain and the subsequent modification of the respective condensation prod...

The broad-host-range plasmid RP4 encodes a highly efficient partitioning function, termed par, that is capable of stabilizing plasmids in a variety of Gram-negative bacteria independently of the nature of the replicon. The mechanism responsible for plasmid stabilization by this locus appears to be a complex system which includes a site-specific recombination system mediating resolution of plasmid multimers. In this report we present a detailed study on this multimer resolution system (mrs). The parA gene encodes two forms of a resolvase capable of catalysing site-specific recombination between specific sites situated in the promoter region of the parCBA operon. The two ParA proteins that are produced as a result of independent translation initiation at two different start codons within the same open reading frame were overexpressed in Escherichia coli and partially purified. Both forms of the enzyme are able to recombine a supercoiled cointegrate substrate containing two cis-acting elements with the same orientation in an in vitro resolution assay. ParA-mediated, site-specific recombination was found to be independent of any other gene product encoded by the RP4 par locus in vitro and in vivo. The DNA-binding sites for the ParA resolvase were determined using DNase I protection experiments. The results identified three binding sites within the mrs cis-acting region. Both the biochemical properties of the ParA protein and the organization of the cis-acting recombination site revealed a high degree of similarity to the site-specific recombination systems of Tn3-like transposable elements suggesting an evolutionary relationship.