Antibiotics blocking bacterial cell wall assembly (beta-lactams and glycopeptides) are facing a challenge from the progressive spread of resistant pathogens. Lantibiotics are promising candidates to alleviate this problem. Microbisporicin, the most potent antibacterial among known comparable lantibiotics, was discovered during a screening applied to uncommon actinomycetes. It is produced by Microbispora sp. as two similarly active and structurally related polypeptides (A1, 2246-Da and A2, 2230-Da) of 24 amino acids linked by 5 intramolecular thioether bridges. Microbisporicin contains two posttranslational modifications that have never been reported previously in lantibiotics: 5-chloro-trypthopan and mono- (in A2) or bis-hydroxylated (in A1) proline. Consistent with screening criteria, microbisporicin selectively blocks peptidoglycan biosynthesis, causing cytoplasmic UDP-linked precursor accumulation. Considering its spectrum of activity and its efficacy in vivo, microbisporicin represents a promising antibiotic to treat emerging infections.

In glycopeptide-resistant enterococci and staphylococci, high-level resistance is achieved by replacing the C-terminal D-alanyl-D-alanine of lipid II with D-alanyl-D-lactate, thus reducing glycopeptide affinity for cell wall targets. Reorganization of the cell wall in these organisms is directed by the vanHAX gene cluster. Similar self-resistance mechanisms have been reported for glycopeptide-producing actinomycetes. We investigated glycopeptide resistance in Nonomuraea sp. ATCC 39727, the producer of the glycopeptide A40926, which is the precursor of the semisynthetic antibiotic dalbavancin, which is currently in phase III clinical trials. The MIC of Nonomuraea sp. ATCC 39727 toward A40926 during vegetative growth was 4 g/ml, but this increased to ca. 20 g/ml during A40926 production. vanHAX gene clusters were not detected in Nonomuraea sp. ATCC 39727 by Southern hybridization or by PCR with degenerate primers. However, the dbv gene cluster for A40926 production contains a gene, vanY ( Actinomycetes are Gram-positive mycelial bacteria with a complex life cycle that consists of vegetative growth followed by the formation of aerial hyphae and ultimately spore formation, the last allowing both dispersal and persistence under unfavorable conditions. The onset of morphological differentiation generally coincides with the production of secondary metabolites, including many antibiotics of immense clinical and commercial importance. Antibiotic-producing actinomycetes must possess mechanisms to avoid suicide by their own toxic products. Several such resistance mechanisms have evolved, including target modification, antibiotic inactivation or sequestration, and efflux mechanisms. Microorganisms produce secondary metabolites mainly during the stationary phase of growth, and resistance genes are often coregulated with those for antibiotic production (8, 28).

Important classes of antibiotics acting on bacterial cell wall biosynthesis, such as beta-lactams and glycopeptides, are used extensively in therapy and are now faced with a challenge because of the progressive spread of resistant pathogens. A discovery program was devised to target novel peptidoglycan biosynthesis inhibitors capable of overcoming these resistance mechanisms. The microbial products were first screened according to their differential activity against Staphylococcus aureus and its L-form. Then, activities insensitive to the addition of a beta-lactamase cocktail or d-alanyl-d-alanine affinity resin were selected. Thirty-five lantibiotics were identified from a library of broth extracts produced by 40,000 uncommon actinomycetes. Five of them showed structural characteristics that did not match with any known microbial metabolite. In this study, we report on the production, structure determination, and biological activity of one of these novel lantibiotics, namely, planosporicin, which is produced by the uncommon actinomycete Planomonospora sp. Planosporicin is a 2194 Da polypeptide originating from 24 proteinogenic amino acids. It contains lanthionine and methyllanthionine amino acids generating five intramolecular thioether bridges. Planosporicin selectively blocks peptidoglycan biosynthesis and causes accumulation of UDP-linked peptidoglycan precursors in growing bacterial cells. On the basis of its mode of action and globular structure, planosporicin can be assigned to the mersacidin (20 amino acids, 1825 Da) and the actagardine (19 amino acids, 1890 Da) subgroup of type B lantibiotics. Considering its spectrum of activity against Gram-positive pathogens of medical importance, including multi-resistant clinical isolates, and its efficacy in vivo, planosporicin represents a potentially new antibiotic to treat emerging pathogens.

dGlycopeptides and ␤-lactams inhibit bacterial peptidoglycan synthesis in Gram-positive bacteria; resistance to these antibiotics is studied intensively in enterococci and staphylococci because of their relevance to infectious disease. Much less is known about antibiotic resistance in glycopeptide-producing actinomycetes that are likely to represent the evolutionary source of resistance determinants found in bacterial pathogens. Nonomuraea sp. ATCC 39727, the producer of A40926 (the precursor for the semisynthetic dalbavancin), does not harbor the canonical vanHAX genes. Consequently, we investigated the role of the ␤-lactamsensitive D,D-peptidase/D,D-carboxypeptidase encoded by vanY n , the only van-like gene found in the A40926 biosynthetic gene cluster, in conferring immunity to the antibiotic in Nonomuraea sp. ATCC 39727. Taking advantage of the tools developed recently to genetically manipulate this uncommon actinomycete, we varied vanY n gene dosage and expressed vanH at A at X at from the teicoplanin producer Actinoplanes teichomyceticus in Nonomuraea sp. ATCC 39727. Knocking out vanY n , complementing a vanY n mutant, or duplicating vanY n had no effect on growth but influenced antibiotic resistance and, in the cases of complementation and duplication, antibiotic production. Nonomuraea sp. ATCC 39727 was found to be resistant to penicillins, but its glycopeptide resistance was diminished in the presence of penicillin G, which inhibits VanY n activity. The heterologous expression of vanH at A at X at increased A40926 resistance in Nonomuraea sp. ATCC 39727 but did not increase antibiotic production, indicating that the level of antibiotic production is not directly determined by the level of resistance. The vanY n -based self-resistance in Nonomuraea sp. ATCC 39727 resembles the glycopeptide resistance mechanism described recently in mutants of Enterococcus faecium selected in vitro for high-level resistance to glycopeptides and penicillins.

Protoplast preparation, regeneration and fusion represent essential tools for those poorly studied biotechnologically valuable microorganisms inapplicable with the current molecular biology protocols. The protoplast production and regeneration method developed for Planobispora rosea and using the combination of hen egg-white lysozyme (HEWL) and Streptomyces globisporus mutanolysin was applied to a set of antibiotic-producing filamentous actinomycetes belonging to the Streptosporangiaceae, Micromonosporaceae and Streptomycetaceae. 10 7 -10 9 protoplasts were obtained from 100 ml of culture, after incubation times in the digestion solution ranging from a few hours to 1 or 2 days depending on the strain. The efficiency of protoplast reversion to the normal filamentous growth varied from 0.1 to nearly 50%. Analysis of cell wall peptidoglycan in three representative strains (Nonomuraea sp. ATCC 39727, Actinoplanes teichomyceticus ATCC 31121 and Streptomyces coelicolor A3(2)) has evidenced structural variations in the glycan strand and in the peptide chain, which may account for the different response to cell digestion and protoplast regeneration treatments. Keywords: antibiotics; bacterial cell wall; glycopeptides; protoplast INTRODUCTIONThe so-called rare or uncommon actinomycetes include a group of filamentous actinomycetes other than Streptomyces spp., which are quite difficult to isolate, cultivate and genetically manipulate. 1 Members of these poorly represented and difficult-to-handle group of microorganisms produce valuable antibiotics. However, the study and the following cost-effective exploitation of uncommon actinomycetes have been slow because of the lack of genetic tools, which hamper strain and product improvement. As mobile genetic elements and conjugation systems are poorly characterized in rare actinomycetes, a crucial methodology to achieve their transformation by exogenous DNA or to recombine whole genomes arising from different cell lines (Whole Genome Shuffling-WGS) is based on protoplast manipulation and fusion. Protoplast preparation and regeneration in Streptomyces spp. were originally reported by Okanishi and co-workers. 2 The method developed for streptomycetes was then applied with uneven success to Micromonospora spp. 3 and Brevibacillus spp., 4 but it soon became clear that the protocol used later was species-or even strain-specific. In other industrially valuable actinomycetes, ad hoc techniques have been developed in some cases, 5-8 but methods endowed with a broad applicability and robustness to be applied for DNA recombination and WGS are not available. A possible explanation for the different response to protocols of protoplast