Oxytetracycline hyper-production through targeted genome reduction of Streptomyces rimosus.

Most biosynthetic gene clusters (BGC) encoding the synthesis of important microbial secondary metabolites, such as antibiotics, are either silent or poorly expressed; therefore, to ensure a strong pipeline of novel antibiotics, there is a need to develop rapid and efficient strain development approaches. This study uses comparative genome analysis to instruct rational strain improvement, using Streptomyces rimosus, the producer of the important antibiotic oxytetracycline (OTC) as a model system. Sequencing of the genomes of two industrial strains M4018 and R6-500, developed independently from a common ancestor, identified large DNA rearrangements located at the chromosome end. We evaluated the effect of these genome deletions on the parental S. rimosus Type Strain (ATCC 10970) genome where introduction of a 145 kb deletion close to the OTC BGC in the Type Strain resulted in massive OTC overproduction, achieving titers that were equivalent to M4018 and R6-500. Transcriptome data supported the hypothesis that the reason for such an increase in OTC biosynthesis was due to enhanced transcription of the OTC BGC and not due to enhanced substrate supply. We also observed changes in the expression of other cryptic BGCs; some metabolites, undetectable in ATCC 10970, were now produced at high titers. This study demonstrated for the first time that the main force behind BGC overexpression is genome rearrangement. This new approach demonstrates great potential to activate cryptic gene clusters of yet unexplored natural products of medical and industrial value.IMPORTANCEThere is a critical need to develop novel antibiotics to combat antimicrobial resistance. Streptomyces species are very rich source of antibiotics, typically encoding 20-60 biosynthetic gene clusters (BGCs). However, under laboratory conditions, most are either silent or poorly expressed so that their products are only detectable at nanogram quantities, which hampers drug development efforts. To address this subject, we used comparative genome analysis of industrial Streptomyces rimosus strains producing high titers of a broad spectrum antibiotic oxytetracycline (OTC), developed during decades of industrial strain improvement. Interestingly, large-scale chromosomal deletions were observed. Based on this information, we carried out targeted genome deletions in the native strain S. rimosus ATCC 10970, and we show that a targeted deletion in the vicinity of the OTC BGC significantly induced expression of the OTC BGC, as well as some other silent BGCs, thus suggesting that this approach may be a useful way to identify new natural products.

Effects of Soluble Dietary Fibre on Exercise Performance and Perception of Fatigue in Young Basketball Players.

Research background: In this study, we investigated the effects of soluble dietary fibre on improving neuromuscular and cardiovascular endurance and perception of fatigue in a closely monitored group of basketball players. Prebiotics have been sidelined in sports nutrition and their effect on performance remains poorly investigated and understood. Experimental approach: Eighteen healthy male basketball players were divided into two groups; one received 17 g/day of soluble dietary fibre (Nutriose®) for four weeks and the other group received placebo. Their morphological characteristics, neuromuscular and cardiovascular endurance, and rating of perceived exertion according to the rating of perceived exertion (RPE) scale were assessed. Measurements were taken before supplementation and after four weeks of supplementation. Faecal samples were collected from all participants immediately before and after the supplementation period, their total DNA extracted and sent for amplicon sequencing. Results and conclusions: In this study, fibre had no statistically significant effect on the vertical-type explosive power, no statistically significant effect on sprint-type explosive power, nor on aerobic and anaerobic endurance in the experimental group. Soluble fibre had a statistically significant effect on reducing the rating of perceived exertion of basketball players during the competitive part of the season (RPE 7.27±0.04 versus 8.82±0.81). This was confirmed by two-way ANOVA with replication, which showed that within-group interaction (p=0.0193), before and after dietary intake (p=0.0049), and between-group interaction before and after dietary intake (p=0.0313) had a significant effect on the result. The overall conclusion of the study is that soluble dietary fibre supplementation does not improve neuromuscular and cardiovascular endurance over a 4-week period. However, fibre supplementation could have a significant effect on reducing the rating of perceived exertion, as shown by the statistics. Both amplicon sequencing and subsequent bioinformatics results suggest that this could be the result of the beneficial effect on the intestinal microbiota and its metabolites. Novelty and scientific contribution: This work highlights the importance of prebiotics in sports nutrition. Dietary fibre has been a neglected component of sports nutrition. This study demonstrated a statistically significant positive effect on the perception of fatigue, highlighting the need for further studies in this direction.

Current Viewpoint on Female Urogenital Microbiome-The Cause or the Consequence?

An increasing amount of evidence implies that native microbiota is a constituent part of a healthy urinary tract (UT), making it an ecosystem on its own. What is still not clear is whether the origin of the urinary microbial community is the indirect consequence of the more abundant gut microbiota or a more distinct separation exists between these two systems. Another area of uncertainty is the existence of a link between the shifts in UT microbial composition and both the onset and persistence of cystitis symptoms. Cystitis is one of the most common reasons for antimicrobial drugs prescriptions in primary and secondary care and an important contributor to the problem of antimicrobial resistance. Despite this fact, we still have trouble distinguishing whether the primary cause of the majority of cystitis cases is a single pathogen overgrowth or a systemic disorder affecting the entire urinary microbiota. There is an increasing trend in studies monitoring changes and dynamics of UT microbiota, but this field of research is still in its infancy. Using NGS and bioinformatics, it is possible to obtain microbiota taxonomic profiles directly from urine samples, which can provide a window into microbial diversity (or the lack of) underlying each patient's cystitis symptoms. However, while microbiota refers to the living collection of microorganisms, an interchangeably used term microbiome referring to the genetic material of the microbiota is more often used in conjunction with sequencing data. It is this vast amount of sequences, which are truly "Big Data", that allow us to create models that describe interactions between different species contributing to an UT ecosystem, when coupled with machine-learning techniques. Although in a simplified predator-prey form these multi-species interaction models have the potential to further validate or disprove current beliefs; whether it is the presence or the absence of particular key players in a UT microbial ecosystem, the exact cause or consequence of the otherwise unknown etiology in the majority of cystitis cases. These insights might prove to be vital in our ongoing struggle against pathogen resistance and offer us new and promising clinical markers.

Reference-Grade Genome and Large Linear Plasmid of Streptomyces rimosus: Pushing the Limits of Nanopore Sequencing.

Streptomyces rimosus ATCC 10970 is the parental strain of industrial strains used for the commercial production of the important antibiotic oxytetracycline. As an actinobacterium with a large linear chromosome containing numerous long repeat regions, high GC content, and a single giant linear plasmid (GLP), these genomes are challenging to assemble. Here, we apply a hybrid sequencing approach relying on the combination of short- and long-read next-generation sequencing platforms and whole-genome restriction analysis by using pulsed-field gel electrophoresis (PFGE) to produce a high-quality reference genome for this biotechnologically important bacterium. By using PFGE to separate and isolate plasmid DNA from chromosomal DNA, we successfully sequenced the GLP using Nanopore data alone. Using this approach, we compared the sequence of GLP in the parent strain ATCC 10970 with those found in two semi-industrial progenitor strains, R6-500 and M4018. Sequencing of the GLP of these three S. rimosus strains shed light on several rearrangements accompanied by transposase genes, suggesting that transposases play an important role in plasmid and genome plasticity in S. rimosus. The polished annotation of secondary metabolite biosynthetic pathways compared to metabolite analysis in the ATCC 10970 strain also refined our knowledge of the secondary metabolite arsenal of these strains. The proposed methodology is highly applicable to a variety of sequencing projects, as evidenced by the reliable assemblies obtained. IMPORTANCE The genomes of Streptomyces species are difficult to assemble due to long repeats, extrachromosomal elements (giant linear plasmids [GLPs]), rearrangements, and high GC content. To improve the quality of the S. rimosus ATCC 10970 genome, producer of oxytetracycline, we validated the assembly of GLPs by applying a new approach to combine pulsed-field gel electrophoresis separation and GLP isolation and sequenced the isolated GLP with Oxford Nanopore technology. By examining the sequenced plasmids of ATCC 10970 and two industrial progenitor strains, R6-500 and M4018, we identified large GLP rearrangements. Analysis of the assembled plasmid sequences shed light on the role of transposases in genome plasticity of this species. The new methodological approach developed for Nanopore sequencing is highly applicable to a variety of sequencing projects. In addition, we present the annotated reference genome sequence of ATCC 10970 with a detailed analysis of the biosynthetic gene clusters.

Modeling of Urinary Microbiota Associated With Cystitis.

A decade ago, when the Human Microbiome Project was starting, urinary tract (UT) was not included because the bladder and urine were considered to be sterile. Today, we are presented with evidence that healthy UT possesses native microbiota and any major event disrupting its "equilibrium" can impact the host also. This dysbiosis often leads to cystitis symptoms, which is the most frequent lower UT complaint, especially among women. Cystitis is one of the most common causes of antimicrobial drugs prescriptions in primary and secondary care and an important contributor to the problem of antimicrobial resistance. Despite this fact, we still have trouble distinguishing whether the primary cause of majority of cystitis cases is a single pathogen overgrowth, or a systemic disorder affecting entire UT microbiota. There are relatively few studies monitoring changes and dynamics of UT microbiota in cystitis patients, making this field of research still an unknown. In this study variations to the UT microbiota of cystitis patients were identified and microbial dynamics has been modeled. The microbial genetic profile of urine samples from 28 patients was analyzed by 16S rDNA Illumina sequencing and bioinformatics analysis. One patient with bacterial cystitis symptoms was prescribed therapy based on national guideline recommendations on antibacterial treatment of urinary tract infections (UTI) and UT microbiota change was monitored by 16S rDNA sequencing on 24 h basis during the entire therapy duration. The results of sequencing implied that a particular class of bacteria is associated with majority of cystitis cases in this study. The contributing role of this class of bacteria - Gammaproteobacteria, was further predicted by generalized Lotka-Volterra modeling (gLVM). Longitudinal microbiota insight obtained from a single patient under prescribed antimicrobial therapy revealed rapid and extensive changes in microbial composition and emphasized the need for current guidelines revision in regards to therapy duration. Models based on gLVM indicated protective role of two taxonomic classes of bacteria, Actinobacteria and Bacteroidia class, which appear to actively suppress pathogen overgrowth.

Identification of pathogens from native urine samples by MALDI-TOF/TOF tandem mass spectrometry.

BACKGROUND: Reliable high-throughput microbial pathogen identification in human urine samples is crucial for patients with cystitis symptoms. Currently employed methods are time-consuming and could lead to unnecessary or inadequate antibiotic treatment. Purpose of this study was to assess the potential of mass spectrometry for uropathogen identification from a native urine sample. METHODS: In total, 16 urine samples having more than 105 CFU/mL were collected from clinical outpatients. These samples were analysed using standard urine culture methods, followed by 16S rRNA gene sequencing serving as control and here described culture-independent MALDI-TOF/TOF MS method being tested. RESULTS: Here we present advantages and disadvantages of bottom-up proteomics, using MALDI-TOF/TOF tandem mass spectrometry, for culture-independent identification of uropathogens (e.g. directly from urine samples). The direct approach provided reliable identification of bacteria at the genus level in monobacterial samples. Taxonomic identifications obtained by proteomics were compared both to standard urine culture test used in clinics and genomic test based on 16S rRNA sequencing. CONCLUSIONS: Our findings indicate that mass spectrometry has great potential as a reliable high-throughput tool for microbial pathogen identification in human urine samples. In this case, the MALDI-TOF/TOF, was used as an analytical tool for the determination of bacteria in urine samples, and the results obtained emphasize high importance of storage conditions and sample preparation method impacting reliability of MS2 data analysis. The proposed method is simple enough to be utilized in existing clinical settings and is highly suitable for suspected single organism infectious etiologies. Further research is required in order to identify pathogens in polymicrobial urine samples.

The functional capacity of plantaricin-producing Lactobacillus plantarum SF9C and S-layer-carrying Lactobacillus brevis SF9B to withstand gastrointestinal transit.

BACKGROUND: We evaluated the functional capacity of plantaricin-producing Lactobacillus plantarum SF9C and S-layer-carrying Lactobacillus brevis SF9B to withstand gastrointestinal transit and to compete among the gut microbiota in vivo. Considering the probiotic potential of Lb. brevis SF9B, this study aims to investigate the antibacterial activity of Lb. plantarum SF9C and their potential for in vivo colonisation in rats, which could be the basis for the investigation of their synergistic functionality. RESULTS: A plantaricin-encoding cluster was identified in Lb. plantarum SF9C, a strain which efficiently inhibited the growth of Listeria monocytogenes ATCC® 19111™ and Staphylococcus aureus 3048. Homology-based three-dimensional (3D) structures of SF9C plantaricins PlnJK and PlnEF were predicted using SWISS-MODEL workspace and the helical wheel representations of the plantaricin peptide helices were generated by HELIQUEST. Contrary to the plantaricin-producing SF9C strain, the S-layer-carrying SF9B strain excluded Escherichia coli 3014 and Salmonella enterica serovar Typhimurium FP1 from the adhesion to Caco-2 cells. Finally, PCR-DGGE analysis of the V2-V3 regions of the 16S rRNA gene confirmed the transit of the two selected lactobacilli through the gastrointestinal tract (GIT). Microbiome profiling via the Illumina MiSeq platform revealed the prevalence of Lactobacillus spp. in the gut microbiota of the Lactobacillus-treated rats, even on the 10th day after the Lactobacillus application, compared to the microbiota of the healthy and AlCl3-exposed rats before Lactobacillus treatment. CONCLUSION: The combined application of Lb. plantarum SF9C and Lb. brevis SF9B was able to influence the intestinal microbiota composition in rats, which was reflected in the increased abundance of Lactobacillus genus, but also in the altered abundances of other bacterial genera, either in the model of healthy or aberrant gut microbiota of rats. The antibacterial activity and capacity to withstand in GIT conditions contributed to the functional aspects of SF9C and SF9B strains that could be incorporated in the probiotic-containing functional foods with a possibility to positively modulate the gut microbiota composition.

Bioprospecting for Genes Encoding Hydrocarbon-Degrading Enzymes from Metagenomic Samples Isolated from Northern Adriatic Sea Sediments.

Three metagenomic libraries were constructed using surface sediment samples from the northern Adriatic Sea. Two of the samples were taken from a highly polluted and an unpolluted site respectively. The third sample from a polluted site had been enriched using crude oil. The results of the metagenome analyses were incorporated in the REDPET relational database (http://redpet.bioinfo.pbf.hr/REDPET), which was generated using the previously developed MEGGASENSE platform. The database includes taxonomic data to allow the assessment of the biodiversity of metagenomic libraries and a general functional analysis of genes using hidden Markov model (HMM) profiles based on the KEGG database. A set of 22 specialised HMM profiles was developed to detect putative genes for hydrocarbon-degrading enzymes. Use of these profiles showed that the metagenomic library generated after selection on crude oil had enriched genes for aerobic n-alkane degradation. The use of this system for bioprospecting was exemplified using potential alkB and almA genes from this library.

Isolation and Characterisation of L. plantarum O1 Producer of Plantaricin as Potential Starter Culture for the Biopreservation of Aquatic Food Products.

Lactobacillus plantarum O1 was isolated from the gut of sea bream (Sparus aurata) and identified with the API biochemical test and MALDI-TOF MS. This strain was further characterised according to the selection criteria for lactic acid bacteria as starter cultures for aquatic food production. L. plantarum O1 showed good antimicrobial activity against pathogenic test microorganisms. Further investigation confirmed it as the producer of the bacteriocin plantaricin. This strain also showed good growth at a wide range of temperatures (from 4 to 45 °C) and a wide range of pH (2-12), even in the presence of 3.5% NaCl. Its viability was also good after lyophilisation and in simulated gastric and small intestinal juice. The strain is a promising probiotic, and our further research will focus on its application in the biopreservation of fresh fish and shellfish.

MEGGASENSE - The Metagenome/Genome Annotated Sequence Natural Language Search Engine: A Platform for the Construction of Sequence Data Warehouses.

The MEGGASENSE platform constructs relational databases of DNA or protein sequences. The default functional analysis uses 14 106 hidden Markov model (HMM) profiles based on sequences in the KEGG database. The Solr search engine allows sophisticated queries and a BLAST search function is also incorporated. These standard capabilities were used to generate the SCATT database from the predicted proteome of Streptomyces cattleya. The implementation of a specialised metagenome database (AMYLOMICS) for bioprospecting of carbohydrate-modifying enzymes is described. In addition to standard assembly of reads, a novel 'functional' assembly was developed, in which screening of reads with the HMM profiles occurs before the assembly. The AMYLOMICS database incorporates additional HMM profiles for carbohydrate-modifying enzymes and it is illustrated how the combination of HMM and BLAST analyses helps identify interesting genes. A variety of different proteome and metagenome databases have been generated by MEGGASENSE.

Gene duplications are extensive and contribute significantly to the toxic proteome of nematocysts isolated from Acropora digitifera (Cnidaria: Anthozoa: Scleractinia).

BACKGROUND: Gene duplication followed by adaptive selection is a well-accepted process leading to toxin diversification in venoms. However, emergent genomic, transcriptomic and proteomic evidence now challenges this role to be at best equivocal to other processess . Cnidaria are arguably the most ancient phylum of the extant metazoa that are venomous and such provide a definitive ancestral anchor to examine the evolution of this trait. METHODS: Here we compare predicted toxins from the translated genome of the coral Acropora digitifera to putative toxins revealed by proteomic analysis of soluble proteins discharged from nematocysts, to determine the extent to which gene duplications contribute to venom innovation in this reef-building coral species. A new bioinformatics tool called HHCompare was developed to detect potential gene duplications in the genomic data, which is made freely available ( https://github.com/rgacesa/HHCompare ). RESULTS: A total of 55 potential toxin encoding genes could be predicted from the A. digitifera genome, of which 36 (65 %) had likely arisen by gene duplication as evinced using the HHCompare tool and verified using two standard phylogeny methods. Surprisingly, only 22 % (12/55) of the potential toxin repertoire could be detected following rigorous proteomic analysis, for which only half (6/12) of the toxin proteome could be accounted for as peptides encoded by the gene duplicates. Biological activities of these toxins are dominatedby putative phospholipases and toxic peptidases. CONCLUSIONS: Gene expansions in A. digitifera venom are the most extensive yet described in any venomous animal, and gene duplication plays a significant role leading to toxin diversification in this coral species. Since such low numbers of toxins were detected in the proteome, it is unlikely that the venom is evolving rapidly by prey-driven positive natural selection. Rather we contend that the venom has a defensive role deterring predation or harm from interspecific competition and overgrowth by fouling organisms. Factors influencing translation of toxin encoding genes perhaps warrants more profound experimental consideration.

Bacterial diversity of polluted surface sediments in the northern Adriatic Sea.

Samples were collected from sea sediments at seven sites in the northern Adriatic Sea that included six sites next to industrial complexes and one from a tourist site (recreational beach). The samples were assayed for alkanes and polycyclic aromatic hydrocarbons. The composition of the hydrocarbon samples suggested that industrial pollution was present in most cases. A sample from one site was also grown aerobically under crude oil enrichment in order to evaluate the response of indigenous bacterial populations to crude oil exposure. Analysis of 16S rRNA gene sequences showed varying microbial biodiversity depending on the level of pollution--ranging from low (200 detected genera) to high (1000+ genera) biodiversity, with lowest biodiversity observed in polluted samples. This indicated that there was considerable biodiversity in all sediment samples but it was severely restricted after exposure to crude oil selection pressure. Phylogenetic analysis of putative alkB genes showed high evolutionary diversity of the enzymes in the samples and suggested great potential for bioremediation and bioprospecting. The first systematic analysis of bacterial communities from sediments of the northern Adriatic Sea is presented, and it will provide a baseline assessment that may serve as a reference point for ecosystem changes and hydrocarbon degrading potential--a potential that could soon gain importance due to plans for oil exploitation in the area.

Combinations of long peptide sequence blocks can be used to describe toxin diversification in venomous animals.

An important mechanism for the evolution of toxins in venomous animals is believed to be the acquisition of genes encoding proteins that switch from physiological to toxic roles following gene duplication. The 'reverse recruitment' hypothesis pertains that these genes can also revert back to physiological functions, although such events are thought to be rare. A non-supervised homology searching method was developed which allowed the peptide diversity of animal toxins to be described as combinations between limited numbers of amino-acid sequence blocks we called 'tox-bits'. Taking the phospholipase A2 (PLA2) protein family as an example, a Bernoulli Trial was used to test if 'tox-bits' were robust enough to distinguish between peptides with physiological or toxin functions. The analysis revealed that discrimination was indeed possible, and supports the very recent 'restriction' hypothesis whereby genes with the potential to encode toxic functions have likely been independently recruited into venom systems and therefore require few, if any, reverse recruitment events. The development of 'tox-bits' provides a novel bioinformatics tool to allow recognition of toxins from other proteins in genome sequences, facilitating the study of gene recruitment and duplication strategies in venom diversification. The 'tox-bits' library is freely available at http://bioserv.pbf.hr/blocks.zip.

Proteomics links the redox state to calcium signaling during bleaching of the scleractinian coral Acropora microphthalma on exposure to high solar irradiance and thermal stress.

Shipboard experiments were each performed over a 2 day period to examine the proteomic response of the symbiotic coral Acropora microphthalma exposed to acute conditions of high temperature/low light or high light/low temperature stress. During these treatments, corals had noticeably bleached. The photosynthetic performance of residual algal endosymbionts was severely impaired but showed signs of recovery in both treatments by the end of the second day. Changes in the coral proteome were determined daily and, using recently available annotated genome sequences, the individual contributions of the coral host and algal endosymbionts could be extracted from these data. Quantitative changes in proteins relevant to redox state and calcium metabolism are presented. Notably, expression of common antioxidant proteins was not detected from the coral host but present in the algal endosymbiont proteome. Possible roles for elevated carbonic anhydrase in the coral host are considered: to restore intracellular pH diminished by loss of photosynthetic activity, to indirectly limit intracellular calcium influx linked with enhanced calmodulin expression to impede late-stage symbiont exocytosis, or to enhance inorganic carbon transport to improve the photosynthetic performance of algal symbionts that remain in hospite. Protein effectors of calcium-dependent exocytosis were present in both symbiotic partners. No caspase-family proteins associated with host cell apoptosis, with exception of the autophagy chaperone HSP70, were detected, suggesting that algal loss and photosynthetic dysfunction under these experimental conditions were not due to host-mediated phytosymbiont destruction. Instead, bleaching occurred by symbiont exocytosis and loss of light-harvesting pigments of algae that remain in hospite. These proteomic data are, therefore, consistent with our premise that coral endosymbionts can mediate their own retention or departure from the coral host, which may manifest as "symbiont shuffling" of Symbiodinium clades in response to environmental stress.

Genome Sequences of the Oxytetracycline Production Strain Streptomyces rimosus R6-500 and Two Mutants with Chromosomal Rearrangements.

The genome sequence of Streptomyces rimosus R6-500, an industrially improved strain which produces high titers of the important antibiotic oxytetracycline, is reported, as well as the genome sequences of two derivatives arising due to the genetic instability of the strain.

Genome Sequence of Streptomyces olindensis DAUFPE 5622, Producer of the Antitumoral Anthracycline Cosmomycin D.

Streptomyces olindensis DAUFPE 5622, which was isolated from a Brazilian soil sample, produces the antitumor anthracycline cosmomycin D. The genome sequence is 9.4 Mb in length, with a G+C content of 71%. Thirty-four putative secondary metabolite biosynthetic gene clusters were identified, including the cosmomycin D cluster.

Predicting substrate specificity of adenylation domains of nonribosomal peptide synthetases and other protein properties by latent semantic indexing.

Successful genome mining is dependent on accurate prediction of protein function from sequence. This often involves dividing protein families into functional subtypes (e.g., with different substrates). In many cases, there are only a small number of known functional subtypes, but in the case of the adenylation domains of nonribosomal peptide synthetases (NRPS), there are >500 known substrates. Latent semantic indexing (LSI) was originally developed for text processing but has also been used to assign proteins to families. Proteins are treated as ''documents'' and it is necessary to encode properties of the amino acid sequence as ''terms'' in order to construct a term-document matrix, which counts the terms in each document. This matrix is then processed to produce a document-concept matrix, where each protein is represented as a row vector. A standard measure of the closeness of vectors to each other (cosines of the angle between them) provides a measure of protein similarity. Previous work encoded proteins as oligopeptide terms, i.e. counted oligopeptides, but used no information regarding location of oligopeptides in the proteins. A novel tokenization method was developed to analyze information from multiple alignments. LSI successfully distinguished between two functional subtypes in five well-characterized families. Visualization of different ''concept'' dimensions allows exploration of the structure of protein families. LSI was also used to predict the amino acid substrate of adenylation domains of NRPS. Better results were obtained when selected residues from multiple alignments were used rather than the total sequence of the adenylation domains. Using ten residues from the substrate binding pocket performed better than using 34 residues within 8 Å of the active site. Prediction efficiency was somewhat better than that of the best published method using a support vector machine.

Evolutionary concepts in natural products discovery: what actinomycetes have taught us.

Actinomycetes are a very important source of natural products for the pharmaceutical industry and other applications. Most of the strains belong to Streptomyces or related genera, partly because they are particularly amenable to growth in the laboratory and industrial fermenters. It is unlikely that chemical synthesis can fulfil the needs of the pharmaceutical industry for novel compounds so there is a continuing need to find novel natural products. An evolutionary perspective can help this process in several ways. Genome mining attempts to identify secondary metabolite biosynthetic clusters in DNA sequences, which are likely to produce interesting chemical entities. There are often technical problems in assembling the DNA sequences of large modular clusters in genome and metagenome projects, which can be overcome partially using information about the evolution of the domain sequences. Understanding the evolutionary mechanisms of modular clusters should allow simulation of evolutionary pathways in the laboratory to generate novel compounds.

Draft Genome Sequence of Streptomyces rapamycinicus Strain NRRL 5491, the Producer of the Immunosuppressant Rapamycin.

Streptomyces rapamycinicus strain NRRL 5491 produces the important drug rapamycin. It has a large genome of 12.7 Mb, of which over 3 Mb consists of 48 secondary metabolite biosynthesis clusters.