Stress-activated expression of a Streptomyces pristinaespiralis multidrug resistance gene (ptr) in various Streptomyces spp. and Escherichia coli

A promoter which controls expression of the pristinamycin multidrug resistance gene ( ptr ) in Streptomyces pristinaspiralis could be induced by physiological stresses in both Streptomyces spp. and Escherichia coli . In S. pristinaspiralis , the ptr promoter ( Pptr ) was induced by pristinamycin I (PI) or pristinamycin II (PII). Streptomyces lividans was adopted as a convenient heterologous host for studies of Pptr regulation since it has no known pristinamycin biosynthetic genes. Two key regulatory features were documented in these studies: many (19 of 70) antibiotics and chemicals with no common targets or structural features induced the Pptr ; induction with PI was most efficient during a transition phase when antibiotic biosynthetic genes are switched on. In Streptomyces coelicolor, Pptr activity was similarly inducible by PI and not dependent on sigma factors HrdA, HrdC, or HrdD. In E. coli, Pptr cloned in the bifunctional promoter probe vector plJ2839 was functional and activated upon entry into stationary phase in the absence of exogenous inducer. Finally, gel-retardation studies demonstrated a Pptr -binding protein in S. lividans (where its activity was PI-inducible), S. coelicolor and S. pristinaespiralis . The fact that this activity was not detected in E. coli suggested the existence of another regulatory system perhaps also present in Streptomyces .     

Pleiotropic functions of a Streptomyces pristinaespiralis autoregulator receptor in development, antibiotic biosynthesis, and expression of a superoxide dismutase

In Streptomyces, a family of related butyrolactones and their corresponding receptor proteins serve as quorum-sensing systems that can activate morphological development and antibiotic biosynthesis. Streptomyces pristinaespiralis contains a gene cluster encoding enzymes and regulatory proteins for the biosynthesis of pristinamycin, a clinically important streptogramin antibiotic complex. One of these proteins, PapR1, belongs to a well known family of Streptomyces antibiotic regulatory proteins. Gel shift assays using crude cytoplasmic extracts detected SpbR, a developmentally regulated protein that bound to the papR1 promoter. SpbR was purified, and its gene was cloned using reverse genetics. spbR encoded a 25-kDa protein similar to Streptomyces autoregulatory proteins of the butyrolactone receptor family, including scbR from Streptomyces coelicolor. In Escherichia coli, purified SpbR and ScbR produced bound sequences immediately upstream of papR1, spbR, and scbR. SpbR DNA-binding activity was inhibited by an extracellular metabolite with chromatographic properties similar to those of the well known gamma-butyrolactone signaling compounds. DNase I protection assays mapped the SpbR-binding site in the papR1 promoter to a sequence homologous to other known butyrolactone autoregulatory elements. A nucleotide data base search showed that these binding motifs were primarily located upstream of genes encoding Streptomyces antibiotic regulatory proteins and butyrolactone receptors in various Streptomyces species. Disruption of the spbR gene in S. pristinaespiralis resulted in severe defects in growth, morphological differentiation, pristinamycin biosynthesis, and expression of a secreted superoxide dismutase.     

Cluster organization of the genes of Streptomyces pristinaespiralis involved in pristinamycin biosynthesis and resistance elucidated by pulsed-field gel electrophoresis

N. BAMAS-JACQUES, S. LORENZON, P. LACROIX, C. DE SWETSCHIN and J. CROUZET.1999. Streptomyces pristinaespiralis synthesizes pristinamycin, a member of the streptogramin antibiotic family which consists of a mixture of two types of chemically unrelated compounds named pristinamycins I and pristinamycins II. In order to estimate the size of the Strep. pristinaespiralis chromosome and to elucidate the organization of the pristinamycin biosynthetic and resistance genes already identified, it was decided to use the pulsed-field gel electrophoresis technique. Results indicate that the Strep. pristinaespiralis chromosome is linear and about 7580 kb, as previously shown for several other Streptomyces species. By hybridization, it could be shown that the biosynthetic and resistance genes for pristinamycins I and pristinamycins II, except for the multidrug resistance gene ptr , are interspersed and seem to be organized as a single large cluster, covering less than 200 kb corresponding to 2·6% of the total size of the chromosome. The consequences and significance of such a genetic organization are discussed.     

Actinomycin Analogues Containing Pipecolic Acid: Relationship of Structure to Biological Activity

Streptomyces antibioticus synthesizes a mixture of actinomycins which differ at the "imino acid" site of the peptide chains. In the presence of exogenous pipecolic acid, several new actinomycins were synthesized and 70% of the proline in the antibiotic mixture was replaced by the analogue. Three new antibiotics (designated Pip 1alpha, Pip 1beta, and Pip 2) were isolated from culture filtrates, purified, and crystallized. The molar ratio of pipecolic acid to proline was: Pip 1alpha, 1:0; Pip 1beta, 1:1; Pip 2, 2:0. These compounds inhibited the growth and cell division of gram-positive, but not gram-negative, bacteria. The relative inhibitory activity against bacteria, Escherichia coli deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase in vitro, and RNA synthesis in Bacillus subtilis and mouse L-929 cells was: actinomycin IV = Pip 1beta > Pip 2 > Pip 1alpha. Protein synthesis in B. subtilis was less affected, and DNA synthesis was inhibited only at higher concentrations of antibiotic tested. In L cells, DNA formation was reduced less than RNA synthesis, whereas protein synthesis was not blocked under the experimental conditions employed. Kinetic studies with B. subtilis revealed that RNA synthesis was inhibited rapidly followed by an inhibition of protein synthesis. All four antibiotics markedly inhibited the replication of vaccinia virus and reovirus in tissue culture cells, but the production of poliovirus was resistant to the antibiotics. These actinomycins bind to DNA, resulting in an elevation of its T(m) and a decrease in the peak extinction of the actinomycins. The mode of action, as well as the structure-activity relationships among the actinomycins, are discussed relative to a previously proposed model of binding.     

Streptogramin-based gene regulation systems for mammalian cells

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.     

Probing the Molecular Mechanisms for Pristinamycin Yield Enhancement in Streptomyces pristinaespiralis

The mechanisms for the enhancement of pristinamycin production in the high-yielding recombinants of Streptomyces pristinaespiralis obtained by genome shuffling were investigated by quantitative real-time PCR (Q-PCR) and amplified fragment length polymorphism (AFLP) techniques. Q-PCR analysis showed that snaB and snbA involved, respectively, in the biosynthesis of pristinamycins II and I component had more extended high expression in the recombinant than that in the ancestor during fermentation process, indicating their expression changes might be key factors during the biosynthesis of the antibiotic. In addition, the antecedent establishment of the high self-resistance to pristinamycin, because ptr resistance gene started high-level expression ahead of the onset of the antibiotic production in the recombinant, might also lead to the increase of the antibiotics yield. AFLP analysis of these recombinants revealed genome variation of two novel genes, the homologs of AfsR regulatory gene and transposase gene, indicating these two gene variations were probably responsible for yield improvement of pristinamycin. This study provided several potential molecular clues for pristinamycin yield enhancement.     

Identification and functional characterization of phenylglycine biosynthetic genes involved in pristinamycin biosynthesis in Streptomyces pristinaespiralis

Pristinamycin I (PI), a streptogramin type B antibiotic produced by Streptomyces pristinaespiralis, contains the aproteinogenic amino acid l-phenylglycine. Recent sequence analysis led to the identification of a set of putative phenylglycine biosynthetic genes. Successive inactivation of the individual genes resulted in a loss of PI production. Production was restored by supplementation with externally added l-phenylglycine, which demonstrates that these genes are involved in phenylglycine biosynthesis and thus probably disclosing the last essential pristinamycin biosynthetic genes. Finally, a putative pathway for phenylglycine synthesis is proposed.