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The aerobic, Gram-positive, mesophilic Ktedonobacteria strains, Uno17T, SOSP1-1T, 1-9T, 1-30T and 150040T, formed mycelia of irregularly branched filaments, produced spores or sporangia, and numerous secondary metabolite biosynthetic gene clusters. The five strains grew at 15–40 °C (optimally at 30 °C) and pH 4.0–8.0 (optimally at pH 6.0–7.0), and had 7.21–12.67 Mb genomes with 49.7–53.7 mol% G+C content. They shared MK9(H2) as the major menaquinone and C16 : 1-2OH and iso-C17 : 0 as the major cellular fatty acids. Phylogenetic and phylogenomic analyses showed that Uno17T and SOSP1-9T were most closely related to members of the genus Dictyobacter , with 94.43–96.21 % 16S rRNA gene similarities and 72.16–81.56% genomic average nucleotide identity. The strain most closely related to SOSP1-1T and SOSP1-30T was Ktedonobacter racemifer SOSP1-21T, with 91.33 and 98.84 % 16S rRNA similarities, and 75.13 and 92.35% average nucleotide identities, respectively. Strain 150040T formed a distinct clade within the order Ktedonobacterales , showing T as Reticulibacter mediterranei gen. nov., sp. nov. (type strain 150 040T=CGMCC 1.17052T=BCRC 81202T) within the family Reticulibacteraceae fam. nov. in the order Ktedonobacterales ; strain SOSP1-1T as Ktedonospora formicarum gen. nov., sp. nov. (type strain SOSP1-1T=CGMCC 1.17205T=BCRC 81203T) and strain SOSP1-30T as Ktedonobacter robiniae sp. nov. (type strain SOSP1-30T=CGMCC 1.17733T=BCRC 81205T) within the family Ktedonobacteraceae ; strain Uno17T as Dictyobacter arantiisoli sp. nov. (type strain Uno17T=NBRC 113155T=BCRC 81116T); and strain SOSP1-9T as Dictyobacter formicarum sp. nov. (type strain SOSP1-9T=CGMCC 1.17206T=BCRC 81204T) within the family Dictyobacteraceae .

The increasing incidence of infections caused by drug-resistant pathogens requires new efforts for the discovery of novel antibiotics. By screening microbial extracts in an assay aimed at identifying compounds interfering with cell wall biosynthesis, based on differential activity against a Staphylococcus aureus strain and its isogenic l-form, the potent enduracyclinones (1, 2), containing the uncommon amino acid enduracididine linked to a six-ring aromatic skeleton, were discovered from different Nonomuraea strains. The structures of 1 and 2 were established through a combination of derivatizations, oxidative cleavages, and NMR analyses of natural and 13C–15N-labeled compounds. Analysis of the biosynthetic cluster provides the combination of genes for the synthesis of enduracididine and type II polyketide synthases. Enduracyclinones are active against Gram-positive pathogens (especially Staphylococcus spp.), including multi-drug-resistant strains, with minimal inhibitory concentrations in the range of 0....

Natural products have provided many molecules to treat and prevent illnesses in humans, animals and plants. While only a small fraction of the existing microbial diversity has been explored for bioactive metabolites, tens of thousands of molecules have been reported in the literature over the past 80 years. Thus, the main challenge in microbial metabolite screening is to avoid the re-discovery of known metabolites in a cost-effective manner. In this perspective, we report and discuss different approaches used in our laboratory over the past few years, ranging from bioactivity-based screening to looking for metabolic rarity in different datasets to deeply investigating a single Streptomyces strain. Our results show that it is possible to find novel chemistry through a limited screening effort, provided that appropriate selection criteria are in place.

Summary Drug-resistant bacterial pathogens pose an urgent public-health crisis. Here, we report the discovery, from microbial-extract screening, of a nucleoside-analog inhibitor that inhibits bacterial RNA polymerase (RNAP) and exhibits antibacterial activity against drug-resistant bacterial pathogens: pseudouridimycin (PUM). PUM is a natural product comprising a formamidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 6′-amino-pseudouridine. PUM potently and selectively inhibits bacterial RNAP in vitro, inhibits bacterial growth in culture, and clears infection in a mouse model of Streptococcus pyogenes peritonitis. PUM inhibits RNAP through a binding site on RNAP (the NTP addition site) and mechanism (competition with UTP for occupancy of the NTP addition site) that differ from those of the RNAP inhibitor and current antibacterial drug rifampin (Rif). PUM exhibits additive antibacterial activity when co-administered with Rif, exhibits no cross-resistance with Rif, and exhibits a spontaneous resistance rate an order-of-magnitude lower than that of Rif. PUM is a highly promising lead for antibacterial therapy.

There is an urgent need for new antibacterial drugs effective against bacterial pathogens resistant to current drugs1–2. Nucleoside-analog inhibitors (NAIs) of viral nucleotide polymerases have had transformative impact in treatment of HIV3and HCV4. NAIs of bacterial RNA polymerase (RNAP) potentially could have major impact on treatment of bacterial infection, particularly because functional constraints on substitution of RNAP nucleoside triphosphate (NTP) binding sites4-5could limit resistance emergence4-5. Here we report the discovery, from microbial extract screening, of an NAI that inhibits bacterial RNAP and exhibits antibacterial activity against a broad spectrum of drug-sensitive and drug-resistant bacterial pathogens: pseudouridimycin (PUM). PUM is a novel microbial natural product consisting of a formamidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 6'-amino-pseudouridine. PUM potently and selectively inhibits bacterial RNAP in vitro, potently and selectively inhibits bacterial growth in culture, and potently clears infection in a mouse model ofStreptococcus pyogenesperitonitis. PUM inhibits RNAP through a binding site on RNAP (the "i+1" NTP binding site) and mechanism (competition with UTP for occupancy of the "i+1" NTP binding site) that differ from those of the RNAP inhibitor and current antibacterial drug rifampin (Rif). PUM exhibits additive antibacterial activity when co-administered with Rif, exhibits no cross-resistance with Rif, and exhibits a spontaneous resistance rate an order-of-magnitude lower than that of Rif. The results provide the first example of a selective NAI of bacterial RNAP, provide an advanced lead compound for antibacterial drug development, and provide structural information and synthetic routes that enable lead optimization for antibacterial drug development.

There is an increased need for new drug leads to treat diseases in humans, animals and plants. A dramatic example is represented by the need for novel and more effective antibiotics to combat multidrug-resistant microbial pathogens. Natural products represent a major source of approved drugs and still play an important role in supplying chemical diversity, despite a decreased interest by large pharmaceutical companies. Novel approaches must be implemented to decrease the chances of rediscovering the tens of thousands of known natural products. In this review, we present an overview of natural product screening, focusing particularly on microbial products. Different approaches can be implemented to increase the probability of finding new bioactive molecules. We thus present the rationale and selected examples of the use of hypersensitive assays; of accessing unexplored microorganisms, including the metagenome; and of genome mining. We then focus our attention on the technology platform that we are currently using, consisting of approximately 70 000 microbial strains, mostly actinomycetes and filamentous fungi, and discuss about high-quality screening in the search for bioactive molecules. Finally, two case studies are discussed, including the spark that arose interest in the compound: in the case of orthoformimycin, the novel mechanism of action predicted a novel structural class; in the case of NAI-112, structural similarity pointed out to a possible in vivo activity. Both predictions were then experimentally confirmed.

NAI-107, a lantibiotic produced by Microbispora sp. 107891, shows potent activity against multi-drug-resistant bacterial pathogens. It is produced as a complex of related molecules, which is unusual for ribosomally synthesized peptides. Here we describe the identification, characterization, and antibacterial activity of the congeners produced by Microbispora sp. 107891 and by the related Microbispora corallina NRRL 30420. These molecules differ by the presence of two, one, or zero hydroxyl groups at Pro-14, by the presence of a chlorine at Trp-4, and/or by the presence of a sulfoxide on the thioether of the first lanthionine.

Upon high throughput screening of 6700 microbial fermentation extracts, we discovered a compound, designated orthoformimycin, capable of inhibiting protein synthesis in vitro with high efficiency. The molecule, whose structure was elucidated by chemical, spectrometric, and spectroscopic methods, contains an unusual orthoformate moiety (hence the name) and belongs to a novel class of translation inhibitors. This antibiotic does not affect any function of the 30S ribosomal subunit but binds to the 50S subunit causing inhibition of translation elongation and yielding polypeptide products of reduced length. Analysis by fluorescence stopped flow kinetics revealed that EF-G-dependent mRNA translocation is inhibited by orthoformimycin, whereas, surprisingly, translocation of the aminoacyl-tRNA seems to be unaffected. © 2013 American Chemical Society.

Lantibiotics are biologically active peptides produced by Gram-positive bacteria. Starting from fermentation broth extracts preselected from a high-throughput screening program for discovering cell-wall inhibitors, we successfully isolated a new lantibiotic produced by Actinoplanes sp., designated as NAI-802. MS and NMR analysis together with explorative chemistry established that NAI-802 consists of 21 amino acids, 19 of which are identical to those present in the class II lantibiotic actagardine. Interestingly, NAI-802 carries one extra alanine and one extra arginine at the N- and C-termini, respectively. As expected from the overall higher positive charge, NAI-802 was slightly more active than actagardine against staphylococci and streptococci. Further improvement of its antibacterial activity was achieved by adding one additional positive charge through conversion of the C-terminal carboxylate into the corresponding basic amide. NAI-802 thus represents a novel promising candidate for treating Gram-positive infections caused by multidrug-resistant pathogens.

In the search for novel antibiotics, natural products continue to represent a valid source of bioactive molecules. During a program aimed at identifying previously unreported taxa of actinomycetes as potential source of novel compounds, we isolated hundreds of different representatives of a new group, initially designated as 'Alpha' and independently described as Actinoallomurus. We report on a PCR-specific method for the detection of this taxon, on appropriate growth conditions and on a pilot-screening program on 78 strains. The strains produce antibacterial or antifungal compounds at a relatively high frequency. Four strains were characterized in further detail: one produced the aromatic polyketide benanomicin B and its dexylosyl derivative; a second strain produced N-butylbenzenesulfonamide; a third strain was an efficient converter of soymeal isoflavonoids from soymeal constituents; and a fourth strain produced several coumermycin-related aminocoumarins, with coumermycin A2 as the major peak, and with some new congeners as minor components of the complex. These data suggest that Actinoallomurus strains possess several pathways for secondary metabolism and represent an attractive source in the search for novel antibiotics.

Microbial pathogens are becoming increasingly resistant to available treatments, and new antibiotics are badly needed, but the pipeline of compounds under development is scarce. Furthermore, the majority of antibiotics under development are improved derivatives of marketed compounds, which are at best only partially effective against prevailing resistance mechanisms. In contrast, antibiotics endowed with new mechanisms of action are expected to be highly effective against multi-drug resistant pathogens. In this review, examples are provided of new antibiotics classes in late discovery or clinical development, arising from three different avenues: (1) compounds discovered and never brought to market by large pharmaceutical companies; (2) old compounds reanalyzed and rejuvinated with today's tools; and (3) newly discovered molecules. For each compound, we will briefly describe original discovery, mechanism of action, any known resistance, antimicrobial profile, and current status of development.

Two novel Gram-positive-staining, acidophilic strains were isolated from soil samples. Both show typical features of filamentous actinomycetes. On the basis of 16S rRNA gene sequence analysis, the strains are members of the family Micromonosporaceae. The two strains contain hydroxydiaminopimelic acid, glycine, alanine and glutamic acid in the peptidoglycan. Fatty acid profiles clearly differentiate the two strains: cyclohexyl C(17 : 0), i-C(16 : 0) and ai-C(17 : 0) are predominant in Delta1(T), while the major components for Delta3(T) are ai-C(17 : 0) and i-C(16 : 0). The two strains also differ in their major menaquinones, MK-9(H(8), H(4), H(6)) for Delta1(T) and MK-9(H(8), H(6)) for Delta3(T), and in phospholipid patterns; Delta1(T) displays phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, methyl phosphatidylethanolamine and an unknown aminophospholipid, while Delta3(T) also contains minor amounts of several unknown phospholipids in addition to these phospholipids. The whole-cell sugars of both strains are galactose, arabinose and xylose. The G+C content of the DNA is 72.7 mol% for Delta1(T) and 71.9 mol% for Delta3(T). On the basis of chemotaxonomic, physiological and phylogenetic data, we propose Rugosimonospora gen. nov. to accommodate the two strains, with the description of Rugosimonospora acidiphila gen. nov., sp. nov. (the type species; type strain Delta1(T) =DSM 45227(T) =NBRC 104874(T)) and Rugosimonospora africana sp. nov. (type strain Delta3(T) =DSM 45228(T) =NBRC 104875(T)).

We identified an Actinoallomurus strain producing NAI-107, a chlorinated lantibiotic effective against multidrug-resistant Gram-positive pathogens and previously reported from the distantly related genus Microbispora. Inclusion of KBr in the production medium of either the Actinoallomurus or the Microbispora producer readily afforded brominated variants of NAI-107, which were designated as NAI-108. The other post-translational modifications naturally occurring in this lantibiotic family (i.e., hydroxylation of Pro-14 and C-terminal decarboxylation) were unaffected by the presence of a brominated tryptophan. In addition to being the first example of a bromine-containing lantibiotic, NAI-108 displayed a small but consistent improvement in antibacterial activity against all tested strains. The brominated lantibiotic maintained the same rapid bactericidal activity as NAI-107 but at reduced concentrations, consistent with its increased potency and with the role played by the hydrophobicity of the first lanthionine ring. NAI-108 thus represents an interesting addition to a promising family of potent and effective lantibiotics.

Natural products represent a major source of approved drugs and still play an important role in supplying chemical diversity. Consistently, 2014 has seen new, natural product-derived antibiotics approved for human use by the US Food and Drug Administration. One of the recently approved second-generation glycopeptides is dalbavancin, a semi-synthetic derivative of the natural product A40,926. This compound inhibits bacterial growth by binding to lipid intermediate II (Lipid II), a key intermediate in peptidoglycan biosynthesis. Like other recently approved antibiotics, dalbavancin has a complex history of preclinical and clinical development, with several companies contributing to different steps in different years. While our work on dalbavancin development stopped at the previous company, intriguingly our current pipeline includes two more Lipid II-binding natural products or derivatives thereof. In particular, we will focus on the properties of NAI-107 and related lantibiotics, which originated from recent screening and characterization efforts.

Background The filamentous actinomycete Microbispora ATCC-PTA-5024 produces the lantibiotic NAI-107, which is an antibiotic peptide effective against multidrug-resistant Gram-positive bacteria. In actinomycetes, antibiotic production is often associated with a physiological differentiation program controlled by a complex regulatory and metabolic network that may be elucidated by the integration of genomic, proteomic and bioinformatic tools. Accordingly, an extensive evaluation of the proteomic changes associated with NAI-107 production was performed on Microbispora ATCC-PTA-5024 by combining two-dimensional difference in gel electrophoresis, mass spectrometry and gene ontology approaches. Results Microbispora ATCC-PTA-5024 cultivations in a complex medium were characterized by stages of biomass accumulation (A) followed by biomass yield decline (D). NAI-107 production started at 90 h (A stage), reached a maximum at 140 h (D stage) and decreased thereafter. To reveal patterns of differentially represented proteins associated with NAI-107 production onset and maintenance, differential proteomic analyses were carried-out on biomass samples collected: i) before (66 h) and during (90 h) NAI-107 production at A stage; ii) during three time-points (117, 140, and 162 h) at D stage characterized by different profiles of NAI-107 yield accumulation (117 and 140 h) and decrement (162 h). Regulatory, metabolic and unknown-function proteins, were identified and functionally clustered, revealing that nutritional signals, regulatory cascades and primary metabolism shift-down trigger the accumulation of protein components involved in nitrogen and phosphate metabolism, cell wall biosynthesis/maturation, lipid metabolism, osmotic stress response, multi-drug resistance, and NAI-107 transport. The stimulating role on physiological differentiation of a TetR-like regulator, originally identified in this study, was confirmed by the construction of an over-expressing strain. Finally, the possible role of cellular response to membrane stability alterations and of multi-drug resistance ABC transporters as additional self-resistance mechanisms toward the lantibiotic was confirmed by proteomic and confocal microscopy experiments on a Microbispora ATCC-PTA-5024 lantibiotic-null producer strain which was exposed to an externally-added amount of NAI-107 during growth. Conclusion This study provides a net contribution to the elucidation of the regulatory, metabolic and molecular patterns controlling physiological differentiation in Microbispora ATCC-PTA-5024, supporting the relevance of proteomics in revealing protein players of antibiotic biosynthesis in actinomycetes. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2369-z) contains supplementary material, which is available to authorized users.

A novel, Gram-positive bacterial strain was isolated from forest soil. Among species with validly published names, the 16S rRNA gene sequence is related most closely (approx. 93 % similarity) to that of Sporichthya polymorpha DSM 43042T. However, differently from this species, it forms both vegetative and aerial mycelia. The aerial hyphae are straight to slightly flexuous, starting to septate to form chains of more than 20 cylindrical spores with a rugose surface. The strain is acidophilic, with a pH range for robust growth between 4.3 and 6.8 and an optimum around 6.0. The peptidoglycan type is A3γ ll-Dpm–Gly. The polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides and two unknown phospholipids. Predominant menaquinones are MK-9(H6) and -9(H4), and iso- and anteiso-branched C16 : 0 and C17 : 0 are the main cellular fatty acids. The DNA G+C content is 71.9 mol%. The distinct phylogenetic position and the unusual combination of chemotaxonomic characteristics justify the proposal of Catenulispora gen. nov., with the type species Catenulispora acidiphila sp. nov. (type strain, ID139908T =DSM 44928T=NRRL B-24433T). Catenulisporaceae fam. nov. is also proposed.

The discovery of new antibiotics and other bioactive microbial metabolites continues to be an important objective in new drug research. Since extensive screening has led to the discovery of thousands of bioactive microbial molecules, new approaches must be taken in order to reduce the probability of rediscovering known compounds. The authors have recently isolated slow-growing acidophiles belonging to the novel genera Catenulispora and Actinospica within the order Actinomycetales. These strains, which likely belong to a new suborder, grow as filamentous mycelia, have a genome size around 8 Mb, and produce antimicrobial activities. In addition, a single strain harbours simultaneously genes encoding type I and type II polyeketide synthases, as well as non-ribosomal peptide synthetases. The metabolite produced by one strain was identified as a previously reported dimeric isochromanequinone. In addition, at least the Catenulispora strains appear globally distributed, since a PCR-specific signal could be detected in a significant fraction of acidic soils from different continents, and similar strains have been independently isolated from an Australian soil (Jospeh et al., Appl Environ Microbiol 69, 7210-7215, 2003). Thus, these previously uncultured actinomycetes share several features with Streptomyces and related antibiotic-producing genera, and represent a promising source of novel antibiotics.

A novel Gram-positive bacterial strain was isolated from forest soil. According to its 16S rRNA sequence, this strain is a deep-rooting member of the class Actinobacteria. The 16S rRNA sequence is most closely related (approximately 94% identity) to clones of uncultured bacteria detected in different terrestrial environments, while showing only a remote relationship (approximately 90% identity or less) to sequences of cultured species. Cells of the first cultured representative of this phylogenetic cluster are small, short rods that are motile by peritrichous flagella, catalase- and oxidase-positive and grow under aerobic conditions. In liquid culture, flagella from different cells can aggregate to form networks, clearly visible under the light microscope. The peptidoglycan contains meso-diaminopimelic acid and is directly cross-linked (type A1gamma). Mycolic acids are not present. The polar lipids are phosphatidylinositol and an unidentified phospholipid. Menaquinone MK-7(H4) was detected as the predominant isoprenoid quinone. Oleic, 14-methylpentadecanoic, hexadecanoic and omega6c-heptadecenoic acids are the predominant components of the cellular fatty acid profile. The DNA G + C content is 71 mol%. The distinct phylogenetic position and the unusual combination of chemotaxonomic characteristics justify the proposal of a new genus and species, Conexibacter woesei gen. nov., sp. nov., with the type strain ID131577T (=DSM 14684T =JCM 11494T).

We report the draft genome sequence of Microbispora sp. strain ATCC-PTA-5024, a soil isolate that produces NAI-107, a new lantibiotic with the potential to treat life-threatening infections caused by multidrug-resistant Gram-positive pathogens. The draft genome of strain Microbispora sp. ATCC-PTA-5024 consists of 8,543,819 bp, with a 71.2% G+C content and 7,860 protein-coding genes.

Background NAI-107, produced by the actinomycete Microbispora sp. ATCC-PTA-5024, is a promising lantibiotic active against Gram-positive bacteria and currently in late preclinical-phase. Lantibiotics (lanthionine-containing antibiotics) are ribosomally synthesized and post-translationally modified peptides (RiPPs), encoded by structural genes as precursor peptides. The biosynthesis of biologically active compounds is developmentally controlled and it depends upon a variety of environmental stimuli and conditions. Inorganic phosphate (Pi) usually negatively regulates biologically-active molecule production in Actinomycetes, while it has been reported to have a positive control on lantibiotic production in Firmicutes strains. So far, no information is available concerning the Pi effect on lantibiotic biosynthesis in Actinomycetes. Results After having developed a suitable defined medium, Pi-limiting conditions were established and confirmed by quantitative analysis of polyphosphate accumulation and of expression of selected Pho regulon genes, involved in the Pi-limitation stress response. Then, the effect of Pi on Microbispora growth and NAI-107 biosynthesis was investigated in a defined medium containing increasing Pi amounts. Altogether, our analyses revealed that phosphate is necessary for growth and positively influences both growth and NAI-107 production up to a concentration of 5 mM. Higher Pi concentrations were not found to further stimulate Microbispora growth and NAI-107 production. Conclusion These results, on one hand, enlarge the knowledge on Microbispora physiology, and, on the other one, could be helpful to develop a robust and economically feasible production process of NAI-107 as a drug for human use. Electronic supplementary material The online version of this article (doi:10.1186/s12934-014-0133-0) contains supplementary material, which is available to authorized users.

Different analytical techniques were applied to describe the localization of lead and chromium in the tissues of walnut (Juglans regia) and maple (Acer saccharinum) plants exposed to soils that had been artificially contaminated with heavy metals. Two X-ray-based techniques, synchrotron radiation X-ray fluorescence (μ-SRXF) and X-ray microanalysis (SEM/EDX), were utilized in association with induced coupled plasma optical emission spectrometry (ICP-OES). These techniques allowed the definition of maps showing a preferential accumulation of lead in the root periderm of both plants and a limited translocation of the metal to the stems. Accordingly, estimation of the lead concentration with ICP-OES showed that roots contained, on a dry weight basis, 20 to 40 times more metal than stems. Chromium, supplied to the plants as Cr3+ or Cr6+, was taken up only in the latter case and in limited amounts. In general, walnut was more efficient than maple in the uptake of lead and chromium. The combination of X...

Ca.te.nu.li.spo'ra.les. N.L. fem. n. Catenulispora, type genus of the order; suff. -ales, ending to denote an order; N.L. fem. pl. n. Catenulisporales, the Catenulispora order. Actinobacteria / Actinobacteria / Catenulisporales Keywords: Catenulisporales ord. nov.; Catenulispora; Actinobacteria

Among the growing family of ribosomally synthesized, post-translationally modified peptides, particularly intriguing are class III lanthipeptides containing the triamino acid labionin. In the course of a screening program aimed at finding bacterial cell wall inhibitors, we discovered a new lanthipeptide produced by an Actinoplanes sp. The molecule, designated NAI-112, consists of 22 amino acids and contains an N-terminal labionin and a C-terminal methyl-labionin. Unique among lanthipeptides, it carries a 6-deoxyhexose moiety N-linked to a tryptophan residue. Consistently, the corresponding gene cluster encodes, in addition to the LanKC enzyme characteristic of this lanthipeptide class, a glycosyl transferase. Despite possessing weak antibacterial activity, NAI-112 is effective in experimental models of nociceptive pain, reducing pain symptoms in mice in both the formalin and the chronic constriction injury tests. Thus, NAI-112 represents, after the labyrinthopeptins, the second example of a lanthipeptide effective against nociceptive pain. © 2013 American Chemical Society.

There is a need to develop novel antibiotics for treating infections caused by multiresistant pathogens. Notwithstanding a plethora of novel targets and intensive high-throughput screening, conventional chemistry has yet to deliver these badly needed new drugs. Microorganisms have provided many of the existing antibiotics, but there is a general feeling that the large majority of compounds have already been discovered. Novel assays, used to screen common microbes, can provide novel structural scaffolds for antibiotic discovery. However, the highest impact may come from unexplored microbial sources. Fortunately, there is plenty of previously undescribed, antibiotic-producing bacteria in the environment.

New antibiotics are necessary to treat microbial pathogens that are becoming increasingly resistant to available treatment. Despite the medical need, the number of newly approved drugs continues to decline. We offer an overview of the pipeline for new antibiotics at different stages, from compounds in clinical development to newly discovered chemical classes. Consistent with historical data, the majority of antibiotics under clinical development are natural products or derivatives thereof. However, many of them also represent improved variants of marketed compounds, with the consequent risk of being only partially effective against the prevailing resistance mechanisms. In the discovery arena, instead, compounds with promising activities have been obtained from microbial sources and from chemical modification of antibiotic classes other than those in clinical use. Furthermore, new natural product scaffolds have also been discovered by ingenious screening programs. After providing selected examples, we offer our view on the future of antibiotic discovery.

New antibiotics are necessary to treat microbial pathogens that are becoming increasingly resistant to available treatment. Despite the medical need, the number of newly approved drugs continues to decline. We offer an overview of the pipeline for new antibiotics at different stages, from compounds in clinical development to newly discovered chemical classes. Consistent with historical data, the majority of antibiotics under clinical development are natural products or derivatives thereof. However, many of them also represent improved variants of marketed compounds, with the consequent risk of being only partially effective against the prevailing resistance mechanisms. In the discovery arena, instead, compounds with promising activities have been obtained from microbial sources and from chemical modification of antibiotic classes other than those in clinical use. Furthermore, new natural product scaffolds have also been discovered by ingenious screening programs. After providing selected examples, we offer our view on the future of antibiotic discovery.

Actinomycetes play a relevant role in soil ecology and are also of important biotechnological interest as they produce several bioactive metabolites. Within the filamentous actinomycetes, it would be desirable to recognize and characterize environmental samples containing unusual genera. To this end, we have developed selective primer sets for polymerase chain reaction (PCR) amplification of 16S rDNA from the Actinomycetales families Micromonosporaceae, Streptomycetaceae, Streptosporangiaceae and Thermomonosporaceae, and from the genus Dactylosporangium. Each primer set, evaluated on genomic DNA from reference strains, showed high specificity and good sensitivity. After amplification of DNA extracted from soil samples, the sequence of the cloned PCR products confirmed the specific amplification of the desired group of sequences in at least 95% of the clones for each primer set. The application of these primers to environmental samples showed the frequent occurrence of these groups in soil samples and also revealed sequences that can be attributed to new groups of actinomycetes.

The need for novel antibiotics to fight multidrug-resistant pathogens calls for a return to natural product screening, but novel approaches must be implemented to increase the chances of discovering novel compounds. This chapter illustrates strategic considerations and the required ingredients for screening programs: microbial diversity, samples for screening, targets and assays, assay development and implementation, hit identification and follow-up. When appropriate, we highlight the impact that chemical diversity consisting of mixtures of different compounds, amid a large background of known antibiotics, has on the screening process. Examples of detailed procedures are described for strain isolation and preservation, sample preparation, primary and secondary assays, and extract fractionation. While these limited examples are not sufficient to organize a complete screening program, they provide a basis for understanding the details of microbial product screening in the anti-infective field.

The need for novel antibiotics to fight multidrug-resistant pathogens calls for a return to natural product screening, but novel approaches must be implemented to increase the chances of discovering novel compounds. This chapter illustrates strategic considerations and the required ingredients for screening programs: microbial diversity, samples for screening, targets and assays, assay development and implementation, hit identification and follow-up. When appropriate, we highlight the impact that chemical diversity consisting of mixtures of different compounds, amid a large background of known antibiotics, has on the screening process. Examples of detailed procedures are described for strain isolation and preservation, sample preparation, primary and secondary assays, and extract fractionation. While these limited examples are not sufficient to organize a complete screening program, they provide a basis for understanding the details of microbial product screening in the anti-infective field.

Covering: bacterial genome sequences to 2005 and post-genomic literature to June 2006 A total of 223 complete bacterial genomes are analyzed, with 281 citations, for the presence of genes encoding modular polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). We report on the distribution of these systems in different bacterial taxa and, whenever known, the metabolites they synthesize. We also highlight, in the different bacterial lineages, the PKS and NRPS genes and, whenever known, the corresponding products.

Two novel Gram-positive, acidophilic bacterial strains were isolated from forest soil. According to their 16S rRNA gene sequences, these strains are related closely to each other and form a distinct cluster within the order Actinomycetales. They show the typical features of filamentous actinomycetes, with branched vegetative hyphae and production of aerial hyphae. The distinct phylogenetic positions and the combination of chemotaxonomic characteristics of these strains justify the proposal of Actinospica gen. nov. Both strains display 3-hydroxydiaminopimelic acid plus traces of meso-diaminopimelic acid, the phospholipids diphosphatidylglycerol, phosphatidylethanolamine, methylphosphatidylethanolamine and phosphatidylinositol, the predominant cellular fatty acids i-C15 : 0, i-C16 : 0 and ai-C15 : 0 and the whole-cell sugars mannose and rhamnose. They differ in the fatty acid profiles, in the quantitative ratios of the major menaquinones MK-9(H4), MK-9(H6) and MK-9(H8) and in the occurrence of additional whole-cell sugars (arabinose and xylose in strain GE134766T and galactose in strain GE134769T). Differences in the phenotypic characteristics and in the 16S rRNA gene sequences suggest the description of two species, Actinospica robiniae gen. nov., sp. nov. (the type species) and Actinospica acidiphila sp. nov., with the type strains GE134769T (=DSM 44927T=NRRL B-24432T) and GE134766T (=DSM 44926T=NRRL B-24431T), respectively. The DNA G+C contents of strains GE134769T and GE134766T are 70.8 and 69.2 mol%, respectively. Due to the large phylogenetic distance from known actinomycete genera, it is proposed to accommodate Actinospica gen. nov. in Actinospicaceae fam. nov. In addition, Catenulisporineae subord. nov. is proposed to harbour Actinospicaceae fam. nov. and the newly proposed family Catenulisporaceae, described in the accompanying paper.

A novel bacterial strain that was isolated from an Italian soil and was designated SOSP1-21 T forms branched mycelia in solid and liquid media and has a filamentous morphology similar to that of some genera belonging to the Actinobacteria . Electron microscopy showed that this organism has a grape-like appearance, resulting from interlacing of spores originating from sporophoric hyphae. Ten strains that are morphologically related to SOSP1-21 T were recovered from soil. Phylogenetic analyses of 16S rRNA gene segments confirmed the relatedness of these strains to SOSP1-21 T and indicated that the newly isolated strains form separate clades in a deeply branching lineage. The closest matches for the 16S rRNA sequences of all the strains (around 79% identity) were matches with representatives of the Chloroflexi , although the affiliation with this division was not supported by high bootstrap values. The strains are mesophilic aerobic heterotrophs and are also capable of growing under microaerophilic conditions. They all stain gram positive. Strain SOSP1-21 T contains ornithine, alanine, glutamic acid, serine, and glycine as the peptidoglycan amino acids. In addition, an unusual level of C16:1 2OH (30%) was found in the cellular fatty acids. The G+C content of SOSP1-21 T genomic DNA is 53.9%, and MK-9(H 2 ) was the only menaquinone detected. All these data suggest that SOSP1-21 T and the related strains may constitute a new division of filamentous, spore-forming, gram-positive bacteria. We propose the name Ktedobacter racemifer gen. nov., sp. nov. for strain SOSP1-21 T .

Soil microbes represent an important source of biologically active compounds. These molecules present original and unexpected structure and are selective inhibitors of their molecular targets. At Biosearch Italia, discovery of new bioactive molecules is mostly carried out through the exploitation of a proprietary strain collection of over 50000 strains, mostly unusual genera of actinomycetes and uncommon filamentous fungi. A critical element in a drug discovery based on microbial extracts is the isolation of unexploited groups of microorganisms that are at the same time good producers of secondary metabolites. Molecular genetics can assist in these efforts. We will review the development and application of molecular methods for the detection of uncommon genera of actinomycetes in soil DNA and for the rapid dereplication of actinomycete isolates. The results indicate a substantial presence in many soils of the uncommon genera and a large diversity of isolated actinomycetes. However, while uncommon actinomycete strains may provide an increased chance of yielding novel structures, their genetics and physiology are poorly understood. To speed up their manipulation, we have developed vectors capable of stably maintaining large segments of actinomycete DNA in Escherichia coli and of integrating site specifically in the Streptomyces genome. These vectors are suitable for the reconstruction of gene clusters from smaller segment of cloned DNA, the preparation of large-insert libraries from unusual actinomycete strains and the construction of environmental libraries.

Covering: bacterial genome sequences to 2005 and post-genomic literature to June 2006 A total of 223 complete bacterial genomes are analyzed, with 281 citations, for the presence of genes encoding modular polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). We report on the distribution of these systems in different bacterial taxa and, whenever known, the metabolites they synthesize. We also highlight, in the different bacterial lineages, the PKS and NRPS genes and, whenever known, the corresponding products.

Background PCR amplification of bacterial 16S rRNA genes provides the most comprehensive and flexible means of sampling bacterial communities. Sequence analysis of these cloned fragments can provide a qualitative and quantitative insight of the microbial population under scrutiny although this approach is not suited to large-scale screenings. Other methods, such as denaturing gradient gel electrophoresis, heteroduplex or terminal restriction fragment analysis are rapid and therefore amenable to field-scale experiments. A very recent addition to these analytical tools is represented by microarray technology.