Chemotherapeutic options to treat tuberculosis are severely restricted by the intrinsic resistance of Mycobacterium tuberculosis to the majority of clinically applied antibiotics. Such resistance is partially provided by the low permeability of their unique cell envelope. Here we describe a complementary system that coordinates resistance to drugs that have penetrated the envelope, allowing mycobacteria to tolerate diverse classes of antibiotics that inhibit cytoplasmic targets. This system depends on whiB7, a gene that pathogenic Mycobacterium shares with Streptomyces, a phylogenetically related genus known as the source of diverse antibiotics. In M. tuberculosis, whiB7 is induced by subinhibitory concentrations of antibiotics (erythromycin, tetracycline, and streptomycin) and whiB7 null mutants (Streptomyces and Mycobacterium) are hypersusceptible to antibiotics in vitro. M. tuberculosis is also antibiotic sensitive within a monocyte model system. In addition to antibiotics, whiB7 is induced by exposure to fatty acids that pathogenic Mycobacterium species may accumulate internally or encounter within eukaryotic hosts during infection.Gene expression profiling analyses demonstrate that whiB7 transcription determines drug resistance by activating expression of a regulon including genes involved in ribosomal protection and antibiotic efflux. Components of the whiB7 system may serve as attractive targets for the identification of inhibitors that render M. tuberculosis or multidrug-resistant derivatives more antibioticsensitive.multidrug resistance ͉ Streptomyces ͉ WhiB ͉ microarray ͉ gene expression

Here we describe repressible (PipOFF) as well as inducible (PipON) systems for regulated gene expression in mammalian cells, based on the repressor Pip (pristinamycin-induced protein), which is encoded by the streptogramin resistance operon of Streptomyces coelicolor. Expression of genes placed under control of these systems was responsive to clinically approved antibiotics belonging to the streptogramin group (pristinamycin, virginiamycin, and Synercid). The versatility of these systems was demonstrated by streptogramin-regulated expression of mouse erythropoietin (EPO), human placental secreted alkaline phosphatase (SEAP), or green fluorescent protein (GFP) in diverse cell lines (BHK, CHO, HeLa, and mouse myoblasts). Analysis of isogenic constructs in CHO cells demonstrated the PipOFF system gave lower background and higher induction ratios than the widely used tetracycline-repressible (TetOFF) expression systems. The streptogramin-based expression technology was functionally compatible with the TetOFF system, thus enabling the selective use of different antibiotics to independently control two different gene activities in the same cell.

We identified the thiomuracins, a novel family of thiopeptides produced by a rare-actinomycete bacterium typed as a Nonomuraea species, via a screen for inhibition of growth of the bacterial pathogen Staphylococcus aureus. Thiopeptides are a class of macrocyclic, highly modified peptides that are decorated by thiazoles and defined by a central six-membered heterocyclic ring system. Mining the genomes of thiopeptide-producing strains revealed the elusive biosynthetic route for this class of antibiotics. The thiopeptides are chromosomally encoded, ribosomally synthesized proteins, and isolation of gene clusters for production of thiomuracin and the related thiopeptide GE2270A revealed the post-translational machinery required for maturation. The target of the thiomuracins was identified as bacterial Elongation Factor Tu (EF-Tu). In addition to potently inhibiting a target that is unexploited by marketed human therapeutics, the thiomuracins have a low propensity for selecting for antibiotic resistance and confer no measurable cross-resistance to antibiotics in clinical use.

Pip is a pristinamycin-induced transcriptional regulator protein detected in many Streptomyces species by its ability to specifically bind sequence motifs within the promoter of a Streptomyces pristinaespiralis multidrug resistance gene (ptr). To investigate the possible role of Pip in regulating multidrug resistance, it was purified from a genetically characterized species, Streptomyces coelicolor, utilizing an affinity matrix of the ptr promoter conjugated to magnetic beads. Reverse genetics identified the corresponding locus and confirmed that it encoded Pip, a protein belonging to the TetR family of procaryotic transcriptional repressors. Pip binding motifs were located upstream of the adjacent gene pep, encoding a major facilitator antiporter homologous to ptr. In vivo analysis of antibiotic susceptibility profiles demonstrated that pep conferred elevated levels of resistance only to pristinamycin I (PI), a streptogramin B antibiotic having clinical importance. Purified recombinant Pip was a dimer (in the presence or absence of PI) and displayed a high affinity for its palindromic binding motifs within the ptr promoter and the upstream region of pep. The Pip/ptr promoter complex was dissociated by PI but not by any of the other nonstreptogramin antibiotics that were described previously as transcriptional inducers. These procaryotic regulatory elements served as the basis for the development of systems allowing repression or induction of cloned genes in mammalian and plant cells in response to streptogramin antibiotics (including pristinamycin, virginiamycin, and Synercid®).

Background: WhiB7 is essential for antibiotic resistance in M. tuberculosis. Results: WhiB7 requires conserved residues, including a redox-sensitive center and DNA-binding motif, to coordinate transcription of species-specific drug resistance genes in diverse Actinobacteria. Conclusion: WhiB7 activates species-specific drug resistance genes in Actinobacteria. Significance: Understanding WhiB7 activity may allow the development of drugs that sensitize bacteria to antibiotics.