Antibiotic-producing Microorganisms Research Articles

The Long Scientific Journey of Sirolimus (Rapamycin): From the Soil of Easter Island (Rapa Nui) to Applied Research and Clinical Trials on β-Thalassemia and Other Hemoglobinopathies.

In this review article, we present the fascinating story of rapamycin (sirolimus), a drug able to induce γ-globin gene expression and increased production of fetal hemoglobin (HbF) in erythroid cells, including primary erythroid precursor cells (ErPCs) isolated from β-thalassemia patients. For this reason, rapamycin is considered of great interest for the treatment of β-thalassemia. In fact, high levels of HbF are known to be highly beneficial for β-thalassemia patients. The story of rapamycin discovery began in 1964, with METEI, the Medical Expedition to Easter Island (Rapa Nui). During this expedition, samples of the soil from different parts of the island were collected and, from this material, an antibiotic-producing microorganism (Streptomyces hygroscopicus) was identified. Rapamycin was extracted from the mycelium with organic solvents, isolated, and demonstrated to be very active as an anti-bacterial and anti-fungal agent. Later, rapamycin was demonstrated to inhibit the in vitro cell growth of tumor cell lines. More importantly, rapamycin was found to be an immunosuppressive agent applicable to prevent kidney rejection after transplantation. More recently, rapamycin was found to be a potent inducer of HbF both in vitro using ErPCs isolated from β-thalassemia patients, in vivo using experimental mice, and in patients treated with this compound. These studies were the basis for proposing clinical trials on β-thalassemia patients.

LexR Positively Regulates the LexABC Efflux Pump Involved in Self-Resistance to the Antimicrobial Di-N-Oxide Phenazine in Lysobacter antibioticus.

Myxin, a di-N-oxide phenazine isolated from the soil bacterium Lysobacter antibioticus, exhibits potent activity against various microorganisms and has the potential to be developed as an agrochemical. Antibiotic-producing microorganisms have developed self-resistance mechanisms to protect themselves from autotoxicity. Antibiotic efflux is vital for such protection. Recently, we identified a resistance-nodulation-division (RND) efflux pump, LexABC, involved in self-resistance against myxin in L. antibioticus. Expression of its genes, lexABC, was induced by myxin and was positively regulated by the LysR family transcriptional regulator LexR. The molecular mechanisms, however, have not been clear. Here, LexR was found to bind to the lexABC promoter region to directly regulate expression. Moreover, myxin enhanced this binding. Molecular docking and surface plasmon resonance analysis showed that myxin bound LexR with valine and lysine residues at positions 146 (V146) and 195 (K195), respectively. Furthermore, mutation of K195 in vivo led to downregulation of the gene lexA. These results indicated that LexR sensed and bound with myxin, thereby directly activating the expression of the LexABC efflux pump and increasing L. antibioticus resistance against myxin. IMPORTANCE Antibiotic-producing bacteria exhibit various sophisticated mechanisms for self-protection against their own secondary metabolites. RND efflux pumps that eliminate antibiotics from cells are ubiquitous in Gram-negative bacteria. Myxin is a heterocyclic N-oxide phenazine with potent antimicrobial and antitumor activities produced by the soil bacterium L. antibioticus. The RND pump LexABC contributes to the self-resistance of L. antibioticus against myxin. Herein, we report a mechanism involving the LysR family regulator LexR that binds to myxin and directly activates the LexABC pump. Further study on self-resistance mechanisms could help the investigation of strategies to deal with increasing bacterial antibiotic resistance and enable the discovery of novel natural products with resistance genes as selective markers.

Penicillin-Binding Proteins, β-Lactamases, and β-Lactamase Inhibitors in β-Lactam-Producing Actinobacteria: Self-Resistance Mechanisms.

The human society faces a serious problem due to the widespread resistance to antibiotics in clinical practice. Most antibiotic biosynthesis gene clusters in actinobacteria contain genes for intrinsic self-resistance to the produced antibiotics, and it has been proposed that the antibiotic resistance genes in pathogenic bacteria originated in antibiotic-producing microorganisms. The model actinobacteria Streptomyces clavuligerus produces the β-lactam antibiotic cephamycin C, a class A β-lactamase, and the β lactamases inhibitor clavulanic acid, all of which are encoded in a gene supercluster; in addition, it synthesizes the β-lactamase inhibitory protein BLIP. The secreted clavulanic acid has a synergistic effect with the cephamycin produced by the same strain in the fight against competing microorganisms in its natural habitat. High levels of resistance to cephamycin/cephalosporin in actinobacteria are due to the presence (in their β-lactam clusters) of genes encoding PBPs which bind penicillins but not cephalosporins. We have revised the previously reported cephamycin C and clavulanic acid gene clusters and, in addition, we have searched for novel β-lactam gene clusters in protein databases. Notably, in S. clavuligerus and Nocardia lactamdurans, the β-lactamases are retained in the cell wall and do not affect the intracellular formation of isopenicillin N/penicillin N. The activity of the β-lactamase in S. clavuligerus may be modulated by the β-lactamase inhibitory protein BLIP at the cell-wall level. Analysis of the β-lactam cluster in actinobacteria suggests that these clusters have been moved by horizontal gene transfer between different actinobacteria and have culminated in S. clavuligerus with the organization of an elaborated set of genes designed for fine tuning of antibiotic resistance and cell wall remodeling for the survival of this Streptomyces species. This article is focused specifically on the enigmatic connection between β-lactam biosynthesis and β-lactam resistance mechanisms in the producer actinobacteria.

Spatial Distribution of Antibiotic-Producing Bacteria in Urban Areas. A Case Study in València (Spain).

The emergence of pathogenic bacteria that are multi-resistant to antibiotics lurks in today's society. In the golden age of the discovery of new antibiotic-producing microorganisms, each contribution was a step forward, but currently the progression is no longer so spectacular. The probability of finding new microorganisms and different antibiotics is lower and lower. The use of spatial statistical methods such as the Kriging technique has been shown to be suitable for mapping each parameter, allowing us to determine areas with greater possibilities of locating these microorganisms. For a practical approach of our estimations a total of 12 isolates capable of inhibiting the growth of several control strains (Escherichia coli, Bacillus cereus and at least one other) were analyzed. The isolates were preliminarily characterized, and subsequently identified at the species level by DNA sequence analyses (16S rDNA PCR) and protein analyses (MALDI-TOF MS). Geospatial mapping with RStudio software provide a satisfactory predictive tool for isolation of new microbial isolates.

Resistance-Nodulation-Division Efflux Pump, LexABC, Contributes to Self-Resistance of the Phenazine Di-N-Oxide Natural Product Myxin in Lysobacter antibioticus.

Antibiotic-producing microorganisms have developed several self-resistance mechanisms to protect them from autotoxicity. Transporters belonging to the resistance- nodulation-division (RND) superfamily commonly confer multidrug resistance in Gram-negative bacteria. Phenazines are heterocyclic, nitrogen-containing and redox-active compounds that exhibit diverse activities. We previously identified six phenazines from Lysobacter antibioticus OH13, a soil bacterium emerging as a potential biocontrol agent. Among these phenazines, myxin, a di-N-oxide phenazine, exhibited potent activity against a variety of microorganisms. In this study, we identified a novel RND efflux pump gene cluster, designated lexABC, which is located far away in the genome from the myxin biosynthesis gene cluster. We found a putative LysR-type transcriptional regulator encoding gene lexR, which was adjacent to lexABC. Deletion of lexABC or lexR gene resulted in significant increasing susceptibility of strains to myxin and loss of myxin production. The results demonstrated that LexABC pump conferred resistance against myxin. The myxin produced at lower concentrations in these mutants was derivatized by deoxidation and O-methylation. Furthermore, we found that the abolishment of myxin with deletion of LaPhzB, which is an essential gene in myxin biosynthesis, resulted in significant downregulation of the lexABC. However, exogenous supplementation with myxin to LaPhzB mutant could efficiently induce the expression of lexABC genes. Moreover, lexR mutation also led to decreased expression of lexABC, which indicates that LexR potentially positively modulated the expression of lexABC. Our findings reveal a resistance mechanism against myxin of L. antibioticus, which coordinates regulatory pathways to protect itself from autotoxicity.

ISOLATION OF ANTIBIOTIC PRODUCING BACTERIA FROM POND SOIL, GUDLAVALLERU

Soil being a major reservoir for microorganisms it is a source of interest for isolation of antibiotic producing organisms. The emergence of antibiotic resistance and need for better, broad spectrum antibiotics is always in high demand. In the present study, antibiotic producing bacteria were isolated from a local soil sample. Total ten soil samples were collected from local pond aseptically and subjected to serial dilution. Crowded plate technique was employed for the isolation of the colony. Total five isolated were isolated which exhibited zone of inhibition around the colony. The isolated colonies were subjected to morphological, microscopical and biochemical characterization. All five colonies were found to be gram positive, non-sporulating organisms and found they belong to the Actinobacteria class. The isolated colonies were subjected to screening for antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis and Yeast by perpendicular streak method. The primary screening results conclude that except one colony all have good antimicrobial activity. One colony found to be highly potential activity which had inhibition towards gram positive, gram negative, sporulating and fungal activity. This study may contribute in providing information on the antibiotic producing microorganisms in soil. Further characterization, purification, and structural elucidation are recommended to know the novelty, quality and commercial value of these antibiotics.

Isolation and Molecular Characterization of Antibiotic Producing Bacillus licheniformis Strains Isolated from Soil

Currently, there is an increase prevalence of antibiotic-resistant bacteria worldwide. Therefore, the need for characterization of naturally occuring antibiotics with less antibiotic resistance is required. Soil resources contains valuable antibiotic producing microorganisms that increasingly being utilized for the production of suitable antibiotics. Therefore, this study aimed at identifying an antibiotic bacteria with ability of producing antibiotic that is isolated from soil samples collected from Al Zarqa provenance, an arid area in Jordan. Morphological and biochemical characterization of the isolates were carried out and found that all of the isolates belong to Bacillus genus. Further confirmation of the characterization of the bacteria was done by ribosomal RNA and PCR. The results reveal that the isolates represent Basilluslicheniformis. These bacilli were further investigated for antimicrobial activities against 6 ATCC human pathogens viz., S. aureus, S. pneumonia, Salmonella typhi., E. coli, P. mirabels and E. cloacae. Additionally, the results of Gas Chromatography Mass Spectrometry (GCMS) of ethyl acetate extracts for B. licheniformis secondary metabolites showed that they contain two main antimicrobial compounds namely Pyrrolo [1, 2-a] pyrazine-1, 4-dione,hexahydro and Trans-13-octadecenoic acid. The present work maybe suggests that soil isolates from the studied arid area include antibiotic producing strains that can be utilized commercially.

Effects of carbon and nitrogen sources on the antibacterial activity of Bacillus tequilensis BSM-F symbiotic with Halichondria panicea sponge from the Cabbiya Coast, Madura, Indonesia

Sponges form obligate symbiotic associations with antibiotic-producing microorganisms. Many factors, including climate change, anthropogenic pollution, introduced species, and nutrient enrichment, have all been linked to the specific character of marine-symbiotic microorganisms. This recent study investigated the effects of carbon and nitrogen sources on the antibacterial activity of Bacillus tequilensis BSM-F in solid fermentation media, Zobell Marine Agar (ZMA), ZMA was added with various carbon and nitrogen sources at different concentrations to evaluate the effect of enrichment media on the antibacterial activity. The carbon sources used were glucose, glycerol, lactose, and starch, while the nitrogen sources were beef extract, soybean meal, malt extract, and casein. Each of which was added to ZMA at 0.5%, 1%, 1.5%, and 2% w/v. The inhibitory activity was denoted as the activity index, i.e., the ratio of the diameter of zone of inhibition to the diameter of the bacterial colony. B. tequilensis BSM-F exhibited the highest inhibitory activity against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 when cultured in media containing glycerol and casein. The optimum condition was achieved when glycerol and casein were each added to ZMA at 1.5% w/v.

SCREENING OF MULTI BIOACTIVE METABOLITES PRODUCING MICROORGANISMFROM GARDEN SOIL OF DR. M. G. R EDUCATIONAL AND RESEARCH INSTITUTE, CHENNAI

Objective: Screening of industrial important bioactive metabolites of Antibiotics, hydrolytic enzymes producing microorganism from garden soil of Dr. M. G. R Educational and Research Institute, Chennai.
 Methods: Desired ten soil samples taken and were serially diluted. Crowded plate method used for antibiotic-producing microorganism and Starch agar medium and gelatin medium tests were performed for hydrolytic enzymes (Amylase and Gelatinase).
 Results: Among the tested soil samples, antibiotic producing microorganisms were not found, but has hydrolytic enzymes amylase and gelatinase. Thus screened soil samples biochemically identified as Bacillus species.
 Conclusion: This study concludes that, the collected sample, produced Antibiotic negative result and it’s possess other industrial important hydrolytic enzymes. Thus Screening of more bioactive metabolites producing ability from a single isolate, will be more useful for effective screening.

CytA, a reductase in the cytorhodin biosynthesis pathway, inactivates anthracycline drugs in Streptomyces

Antibiotic-producing microorganism can develop strategies to deal with self-toxicity. Cytorhodins X and Y, cosmomycins A and B, and iremycin, are produced as final products from a marine-derived Streptomyces sp. SCSIO 1666. These C-7 reduced metabolites show reduced antimicrobial and comparable cytotoxic activities relative to their C-7 glycosylated counterparts. However, the biosynthetic mechanisms and relevant enzymes that drive C-7 reduction in cytorhodin biosynthesis have not yet been characterized. Here we report the discovery and characterization of a reductase, CytA, that mediates C-7 reduction of this anthracycline scaffold; CytA endows the producer Streptomyces sp. SCSIO 1666 with a means of protecting itself from the effects of its anthracycline products. Additionally, we identified cosmomycins C and D as two intermediates involved in cytorhodin biosynthesis and we also broadened the substrate specificity of CytA to clinically used anthracycline drugs.

Screening for Antibiotic-Producing Bacteria from Imbak Canyon Conservation Area (ICCA)

Antibiotic resistance is an escalating threat to public health. Therefore, there is an urgent need for new antibiotics. This study aims to screen for antibiotic-producing microorganisms from the forest soil of Batu Timbang. Soil samples were collected, diluted and spread plated onto 1/5 Nutrient Agar (NA) and Actinomycete Isolation Agar (AIA) for the isolation of antibiotic-producing microorganisms. A total of 180 bacterial isolates were screened for their antibiotic-producing ability, and ten were tested positive for inhibitory activity against one or more test pathogens via agar overlay assay (Staphylococcus aureus ATCC BAA-1717, Enterococcus faecalis ATCC 700802, and Acinetobacter baumannii ATCC BAA-1605). Ten bacterial isolates were subjected to 16S rRNA gene amplification and gene sequence analysis. The isolates were identified to be closely related to the genus Variovorax, Streptomyces, Kitasatospora, Chromobacterium, Burkholderia, Pseudomonas and Massilia. Three isolates (Variovorax sp. A5, Variovorax sp. A6 and Kitasatospora sp. H8) are potentially novel as these isolates form a different clade from their respective closely related species via phylogenetic tree analysis using reference sequences obtained from GenBank/EMBL/DDBJ databases. The antibiotics produced by the bacterial isolates might potentially be new, as novel species might possess unique biosynthetic gene clusters to produce new compounds. Nevertheless, further taxonomic identification and antibiotic isolation work is required. This study has revealed the potential of antibiotic discovery from Batu Timbang (Imbak Canyon Conservation Area) and its implications in tackling antibiotic resistance.

Dissolution of the Disparate: Co-ordinate Regulation in Antibiotic Biosynthesis.

Discovering new antibiotics is vital to combat the growing threat of antimicrobial resistance. Most currently used antibiotics originate from the natural products of actinomycete bacteria, particularly Streptomyces species, that were discovered over 60 years ago. However, genome sequencing has revealed that most antibiotic-producing microorganisms encode many more natural products than previously thought. Biosynthesis of these natural products is tightly regulated by global and cluster situated regulators (CSRs), most of which respond to unknown environmental stimuli, and this likely explains why many biosynthetic gene clusters (BGCs) are not expressed under laboratory conditions. One approach towards novel natural product discovery is to awaken these cryptic BGCs by re-wiring the regulatory control mechanism(s). Most CSRs bind intergenic regions of DNA in their own BGC to control compound biosynthesis, but some CSRs can control the biosynthesis of multiple natural products by binding to several different BGCs. These cross-cluster regulators present an opportunity for natural product discovery, as the expression of multiple BGCs can be affected through the manipulation of a single regulator. This review describes examples of these different mechanisms, including specific examples of cross-cluster regulation, and assesses the impact that this knowledge may have on the discovery of novel natural products.

Advances in Antarctic Research for Antimicrobial Discovery: A Comprehensive Narrative Review of Bacteria from Antarctic Environments as Potential Sources of Novel Antibiotic Compounds Against Human Pathogens and Microorganisms of Industrial Importance.

The recent emergence of antibiotic-resistant bacteria has become a critical public health problem. It is also a concern for industries, since multidrug-resistant microorganisms affect the production of many agricultural and food products of economic importance. Therefore, discovering new antibiotics is crucial for controlling pathogens in both clinical and industrial spheres. Most antibiotics have resulted from bioprospecting in natural environments. Today, however, the chances of making novel discoveries of bioactive molecules from various well-known sources have dramatically diminished. Consequently, unexplored and unique environments have become more likely avenues for discovering novel antimicrobial metabolites from bacteria. Due to their extreme polar environment, Antarctic bacteria in particular have been reported as a potential source for new antimicrobial compounds. We conducted a narrative review of the literature about findings relating to the production of antimicrobial compounds by Antarctic bacteria, showing how bacterial adaptation to extreme Antarctic conditions confers the ability to produce these compounds. We highlighted the diversity of antibiotic-producing Antarctic microorganisms, including the phyla Proteobacteria, Actinobacteria, Cyanobacteria, Firmicutes, and Bacteroidetes, which has led to the identification of new antibiotic molecules and supports the belief that research on Antarctic bacterial strains has important potential for biotechnology applications, while providing a better understanding of polar ecosystems.

English

This study aimed at isolating and characterizing antibiotic producing actinomycetes from a specific geographic location Alba’qa, Jordan, with a known soil character, and study the effect of the environmental factors on their antimicrobial activity, in order to relate the actinomycetes flora to the environmental characteristics. Soils of the Alba’qa region are of uniform composition and contain antimicrobial producing microorganisms. Although the initial aim was to isolate organisms of the genera Actinomyces, other organisms such as Aspergillus and other antibiotic producing microorganisms “mostly bacteria” were also isolated. Actinomycetes showed antimicrobial effect on all tested bacteria “Escherichia coli SQ21, Staphylococcus aureus SQ31, Streptococcus pyogenes SQ33, Serratia marcescens SQ22, Staphyloccocus epidermidis SQ32  and Bacillus spp. SQ41” depending on the media they were cultured on and the temperature they were incubated at. It was established that the optimum antibacterial activity is shown when cultivated on GYE medium at 30°C. This made a beneficial first step to find treatments for the diseases caused by the pathogenic bacteria.   Key words: Actinomycetes, antibiotics, environmental factors, soil.

Secondary Metabolism and Interspecific Competition Affect Accumulation of Spontaneous Mutants in the GacS-GacA Regulatory System in Pseudomonas protegens.

ABSTRACTSecondary metabolites are synthesized by many microorganisms and provide a fitness benefit in the presence of competitors and predators. Secondary metabolism also can be costly, as it shunts energy and intermediates from primary metabolism. In Pseudomonas spp., secondary metabolism is controlled by the GacS-GacA global regulatory system. Intriguingly, spontaneous mutations in gacS or gacA (Gac− mutants) are commonly observed in laboratory cultures. Here we investigated the role of secondary metabolism in the accumulation of Gac− mutants in Pseudomonas protegens strain Pf-5. Our results showed that secondary metabolism, specifically biosynthesis of the antimicrobial compound pyoluteorin, contributes significantly to the accumulation of Gac− mutants. Pyoluteorin biosynthesis, which poses a metabolic burden on the producer cells, but not pyoluteorin itself, leads to the accumulation of the spontaneous mutants. Interspecific competition also influenced the accumulation of the Gac− mutants: a reduced proportion of Gac− mutants accumulated when P. protegens Pf-5 was cocultured with Bacillus subtilis than in pure cultures of strain Pf-5. Overall, our study associated a fitness trade-off with secondary metabolism, with metabolic costs versus competitive benefits of production influencing the evolution of P. protegens, assessed by the accumulation of Gac− mutants.

Secondary Metabolite Production from Industrially Relevant Bacteria is Enhanced by Organic Nanofibers.

Streptomycetes are exploited for the production of a wide range of secondary metabolites, including antibiotics. Therefore, both academic and industrial research efforts are focused on enhancing production of these precious metabolites. So far, this has been mostly achieved by classical or recombinant genetic techniques, in association with process optimization for either submerged or solid state fermentation. New cultivation approaches addressing the natural mycelial growth and life cycle would allow the biosynthetic potential of filamentous strains to be much better exploited. We developed a cultivation system for antibiotic-producing microorganisms which involves electrospun organic nanofibers deposited onto agar plates or immersed in liquid media. Dense filamentous networks of branched hyphae formed by bacterial colonies were found to wrapped around the fibers. We analyzed the effects of fibers on growth and antibiotic production in Streptomyces lividans, and found that the actinorhodin, undecylprodigiosin and calcium dependent antibiotic productions were positively modulated, with a two- to sixfold enhancement compared to standard culture conditions. Highlighting the secondary metabolism-promoting role of nanofibers in bacterial cultures, these results open a route to the design of improved culture systems for microorganisms based on organic nanostructures.

Diverse modes of eco-evolutionary dynamics in communities of antibiotic-producing microorganisms

Relationships between antibiotic interactions and diversity are often examined in ecologically stable in silico models, but building in biologically realistic features is found to promote coexistence and more diversity than idealized models.

Multiple transporters are involved in natamycin efflux in Streptomyces chattanoogensis L10.

Antibiotic-producing microorganisms have evolved several self-resistance mechanisms to prevent auto-toxicity. Overexpression of specific transporters to improve the efflux of toxic antibiotics has been found one of the most important and intrinsic resistance strategies used by many Streptomyces strains. Inthis work, two ATP-binding cassette (ABC) transporter-encoding genes located in the natamycin biosynthetic gene cluster, scnA and scnB, were identified as the primary exporter genes for natamycin efflux in Streptomyces chattanoogensis L10. Two other transporters located outside the cluster, a major facilitator superfamily transporter Mfs1 and an ABC transporter NepI/II were found to play a complementary role in natamycin efflux. ScnA/ScnB and Mfs1 also participate in exporting the immediate precursor of natamycin, 4,5-de-epoxynatamycin, which is more toxic to S. chattanoogensis L10 than natamycin. As the major complementary exporter for natamycin efflux, Mfs1 is up-regulated in response to intracellular accumulation of natamycin and 4,5-de-epoxynatamycin, suggesting a key role in the stress response for self-resistance. This article discusses a novel antibiotic-related efflux and response system in Streptomyces, as well as a self-resistance mechanism in antibiotic-producing strains.

A competitive trade-off limits the selective advantage of increased antibiotic production.

In structured environments, antibiotic producing microorganisms can gain a selective advantage by inhibiting nearby competing species1. However, despite their genetic potential2,3, natural isolates often make only small amounts of antibiotics, and laboratory evolution can lead to loss rather than enhancement of antibiotic production4. Here we show that, due to competition with antibiotic resistant cheater cells, increased levels of antibiotic production can actually decrease the selective advantage to producers. Competing fluorescently-labeled Escherichia coli colicin producers with non-producing resistant and sensitive strains on solid media, we found that while producer colonies can greatly benefit from the inhibition of nearby sensitive colonies, this benefit is shared with resistant colonies growing in their vicinity. A simple model, which accounts for such local competitive and inhibitory interactions, suggests that the advantage of producers varies non-monotonically with the amount of production. Indeed, experimentally varying the amount of production shows a peak in selection for producers, reflecting a trade-off between benefit gained by inhibiting sensitive competitors and loss due to an increased contribution to resistant cheater colonies. These results help explain the low level of antibiotic production observed for natural species, and can help direct laboratory evolution experiments selecting for increased or novel production of antibiotics.

Genomic and Secondary Metabolite Analyses of Streptomyces sp. 2AW Provide Insight into the Evolution of the Cycloheximide Pathway.

The dearth of new antibiotics in the face of widespread antimicrobial resistance makes developing innovative strategies for discovering new antibiotics critical for the future management of infectious disease. Understanding the genetics and evolution of antibiotic producers will help guide the discovery and bioengineering of novel antibiotics. We discovered an isolate in Alaskan boreal forest soil that had broad antimicrobial activity. We elucidated the corresponding antimicrobial natural products and sequenced the genome of this isolate, designated Streptomyces sp. 2AW. This strain illustrates the chemical virtuosity typical of the Streptomyces genus, producing cycloheximide as well as two other biosynthetically unrelated antibiotics, neutramycin, and hygromycin A. Combining bioinformatic and chemical analyses, we identified the gene clusters responsible for antibiotic production. Interestingly, 2AW appears dissimilar from other cycloheximide producers in that the gene encoding the polyketide synthase resides on a separate part of the chromosome from the genes responsible for tailoring cycloheximide-specific modifications. This gene arrangement and our phylogenetic analyses of the gene products suggest that 2AW holds an evolutionarily ancestral lineage of the cycloheximide pathway. Our analyses support the hypothesis that the 2AW glutaramide gene cluster is basal to the lineage wherein cycloheximide production diverged from other glutarimide antibiotics. This study illustrates the power of combining modern biochemical and genomic analyses to gain insight into the evolution of antibiotic-producing microorganisms.