Unit: Bacterial Genetics and Differentiation

Director: Mazodier, Philippe

Streptomyces spp. are Gram-positive, filamentous, soil bacteria. Streptomyces are remarkable in terms of the morphological and metabolic differentiation phenomena that they present. Streptomyces spp. generally synthesise a large number of very diverse secondary metabolites, the best known being antibiotics but others have antifungal, anti-parasitic, antiviral, anti-tumoural, immunosuppressant, insecticides or herbicides activity. We study the role of ATP dependent proteases in morphological and physiological differenciation.

Streptomyces spp. are remarkable in terms of the morphological and metabolic differentiation phenomena that they present. During the later stages of their development (aerial mycelium formation, sporulation). Streptomyces spp. generally synthesise a large number of very diverse secondary metabolites, the best known being antibiotics. Streptomyces and closely related genera produce more than 50% of the 200 commercially available antibiotics and most of the 10,000 known antibiotics. Secondary metabolism also produces bioactive compounds that are used as antifungal, anti-parasitic, antiviral, anti-tumoural, or immunosuppressant agents or as insecticides or herbicides.

Increase in the frequency of pathogenic bacteria multi-resistant to antibiotics is prompting the pharmaceutical industry to screen for new antibiotics.

However, it seems that the niches, which should be first screened for bacteria producing new antibiotics, are not exotic places but rather the established Streptomyces collections of the industry. Indeed, one of the surprises brought by the genome sequencing of Streptomyces was the fact that the best-studied species contain a number of cryptic genes clusters involved in secondary metabolism.

Model organism S. coelicolor was extensively studied for the last 40 years and was known to possess four antibiotic pathways. Sequencing revealed that it contains more than 20 clusters coding for the biosynthesis of secondary metabolites. In S. avermitilis, 30 secondary metabolite gene clusters were found, different from those found in S. coelicolor. If unknown genes are present at such extend in the best-studied strains, this should urge us to reassess the Streptomyces collections made during these last 50 years. They should contain an important unexplored reservoir of genetic and metabolic diversity that it would be very valuable to express

In many organisms, key regulators of the cellular cycle are degraded by the ATP-dependent proteases, Clp. The Clp proteins have a complex organization. They include a proteolytic component, ClpP and a regulating ATPase component, ClpA, ClpC or ClpX, which confers the substrate specificity. We study the effect of ATP dependent proteolysis on Streptomyces morphological and physiological differentiation. In Streptomyces, there are 5 ClpP proteins, and 3 associated ATPase: ClpX, ClpC1 and ClpC2.

We showed that the ClpP1 complex is involved in the degradation of transcriptional regulators, which control the clgR and popR regulons. In particular we showed that the degradation of PopR and ClgR depended on the peptide sequence present at the C terminus of these proteins. In addition we showed that ClpP1 also ensured a post-translational control of the ClgR regulon by degrading several members of this regulon, in particular the Lon protease. We undertook a collaboration with G Bucca and C Smith of the University of Surrey, U.K. to characterize all the genes of the ClgR regulon by using DNA chips.

The phenotype of the clpP1 mutant is "bald", it does not make aerial mycelium. We seek to characterize the targets of ClpP1 involved in the control of differentiation.

Therefore we are interested in the revertants of clpP1 mutant. Those are pseudo-revertants as they kept the clpP1 mutation and the phenotypical reversion to wild type results from the acquisition of suppressor mutation. We seek to characterize this mutation. We built a cosmid bank from the genome of the pseudo-revertant in order to complement the clpP1 mutant. We also undertook collaboration with the DNA chips platform of the Pasteur Institute, to compare the expression of genes in the clpP1 mutant and in the clpP1 pseudo-revertant.

In addition we sought to obtain new mutants of clpP1 presenting a phenotypic reversion after in vivo transposition mutagenesis. These experiments enabled us to obtain 3 mutants able to form aerial mycelium, which we are now analyzing.

As well, we studied the ssrA system in Streptomyces. ssrA genes encoding tmRNA with transfer and messenger RNA functions are ubiquitous in bacteria. It led to the production of marked peptides which are degraded by the ClpP proteases. We obtained the first evidences that the tmRNA tagging system (ssrA and cohort smpB) is active in Streptomyces Obtention of ssrA and smpB mutants of S. lividans showed that the ssrA system is dispensable in Streptomyces. Morphologies of the mutants colonies were similar to the wild type, thus tmRNA-mediated tagging does not seem to have, under conditions used, a significant effect in the Streptomyces differentiation

Figure Legend

Antibiotic production in Streptomyces. Diversity of actions: growth inhibition or induction, differentiation inhibition or induction.

Middle: producing strain, Periphery: eight Streptomyces strains

Producing strain was inoculated 72 hours before patching the eight indicative stains.

Keywords: Streptomyces, differentiation, antibiotic production, proteases, chaperones


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