Streptomyces umbrella toxin particles block hyphal growth of competing species.

Streptomyces are a genus of ubiquitous soil bacteria from which the majority of clinically utilized antibiotics derive1. The production of these antibacterial molecules reflects the relentless competition Streptomyces engage in with other bacteria, including other Streptomyces species1,2. Here we show that in addition to small-molecule antibiotics, Streptomyces produce and secrete antibacterial protein complexes that feature a large, degenerate repeat-containing polymorphic toxin protein. A cryo-electron microscopy structure of these particles reveals an extended stalk topped by a ringed crown comprising the toxin repeats scaffolding five lectin-tipped spokes, which led us to name them umbrella particles. Streptomyces coelicolor encodes three umbrella particles with distinct toxin and lectin composition. Notably, supernatant containing these toxins specifically and potently inhibits the growth of select Streptomyces species from among a diverse collection of bacteria screened. For one target, Streptomyces griseus, inhibition relies on a single toxin and that intoxication manifests as rapid cessation of vegetative hyphal growth. Our data show that Streptomyces umbrella particles mediate competition among vegetative mycelia of related species, a function distinct from small-molecule antibiotics, which are produced at the onset of reproductive growth and act broadly3,4. Sequence analyses suggest that this role of umbrella particles extends beyond Streptomyces, as we identified umbrella loci in nearly 1,000 species across Actinobacteria.

Effects of the pleiotropic regulator DasR on lincomycin production in Streptomyces lincolnensis.

The lincoamide antibiotic lincomycin, derived from Streptomyces lincolnensis, is widely used for the treatment of infections caused by gram-positive bacteria. As a common global regulatory factor of GntR family, DasR usually exists as a regulatory factor that negatively regulates antibiotic synthesis in Streptomyces. However, the regulatory effect of DasR on lincomycin biosynthesis in S. lincolnensis has not been thoroughly investigated. The present study demonstrates that DasR functions as a positive regulator of lincomycin biosynthesis in S. lincolnensis, and its overexpression strain OdasR exhibits a remarkable 7.97-fold increase in lincomycin production compared to the wild-type strain. The effects of DasR overexpression could be attenuated by the addition of GlcNAc in the medium in S. lincolnensis. Combined with transcriptome sequencing and RT-qPCR results, it was found that most structural genes in GlcNAc metabolism and central carbon metabolism were up-regulated, but the lincomycin biosynthetic gene cluster (lmb) were down-regulated after dasR knock-out. However, DasR binding were detected with the DasR responsive elements (dre) of genes involved in GlcNAc metabolism pathway through electrophoretic mobility shift assay, while they were not observed in the lmb. These findings will provide novel insights for the genetic manipulation of S. lincolnensis to enhance lincomycin production. KEY POINTS: • DasR is a positive regulator that promotes lincomycin synthesis and does not affect spore production • DasR promotes lincomycin production through indirect regulation • DasR correlates with nutrient perception in S. lincolnensis.

Effects of Streptomyces sp. HU2014 inoculation on wheat growth and rhizosphere microbial diversity under hexavalent chromium stress.

Wheat (Triticum aestivum L.) is a major foodstuff for over 40% of the world's population. However, hexavalent chromium [Cr(VI)] in contaminated soil significantly affects wheat production and its ecological environment. Streptomyces sp. HU2014 was first used to investigate the effects of Cr (VI) stress on wheat growth. We analyzed the Cr(VI) concentration, physicochemical properties of wheat and soil, total Cr content, and microbial community structures during their interactions. HU2014 reduced the toxicity of Cr(VI) and promoted wheat growth by increasing total nitrogen, nitrate nitrogen, total phosphorus, and Olsen-phosphorus in Cr(VI)-contaminated soil. These four soil variables had strong positive effects on two bacterial taxa, Proteobacteria and Bacteroidota, in the HU2014 treatments. In addition, the level of the dominant Proteobacteria positively correlated with the total Cr content in the soil. Among the fungal communities, which had weaker correlations with soil variables compared with bacterial communities, Ascomycota was the most abundant. Our findings suggest that HU2014 can promote the phytoremediation of Cr(VI)-contaminated soil.

Enhanced production of extracellular L-methioninase by entire cell immobilization of Streptomyces MDMMH4 and examination of its utilization as an antioxidant agent.

Streptomyces MDMMH4 cells were immobilized in various matrices with two different techniques for the enhanced and semi-continuous production of extracellular L-methioninase. Of these, agarose was proven to be the most suitable matrix for the immobilization of cells. The optimal agarose concentration was approximately 3% and the initial cell concentration was 150mg/ml (wet cell weight). Agarose-entrapped cells increased the enzyme yield by 21% compared to the highest yield obtained with free cells. Even after twelve successive and efficient fermentation operations, the agarose blocks had good stability. They maintained 69.3% of the enzyme yield obtained in the first cycle. Applying this process on an industrial scale using agarose-entrapped cells, an inexpensive and renewable matrix will allow the stable production of L-methioninase. The purified L-methioninase could be successfully obtained after applying the purification protocol as mentioned in the previous studies. Subsequently, the purified enzyme showed that L- methioninase possessed moderate scavenging activity with high IC50 values of 390.4µg/mL (corresponding to 11.62U/mL). To our knowledge, this is the first report on L-methioninase production by whole-cell immobilization.

Tetrodecadazinone, a novel tetrodecamycin-pyridazinone hybrid with anti-liver fibrosis activity from Streptomyces sp. HU051.

Tetrodecadazinone (1), a novel tetrodecamycin-pyridazinone hybrid possessing a new 1,2-dimethyl-1-(2-methylnonyl)decahydronaphthalene skeleton, and 4-hydroxydihydrotetrodecamycin (2) were separated from a culture of Streptomyces sp. HU051, together with a known compound, dihydrotetrodecamycin (3). Diverse spectroscopic approaches were applied to assign the structures of 1-3, and the structure of 1 was further confirmed by single crystal X-ray diffraction analysis. Compound 1 is the first example of a pyridazinone-containing natural product. Biosynthetically, 1 is proposed to be derived from a Michael addition reaction of a PKS-derived tetrodecamycin and a piperazic-acid-derived pyridazinone. Biological evaluation revealed 1 could reduce the expressions of extracellular matrix proteins (fibronectin and collagen I) and α-smooth muscle actin (α-SMA) in transforming growth factor-ß (TGF-ß1)-activated LX-2 cells. Preliminary mechanism study showed 1 exerted its anti-liver fibrosis effect by regulating TGF-ß1/Smad2/3 signaling pathway.

Heat Shock Repressor HspR Directly Controls Avermectin Production, Morphological Development, and H2O2 Stress Response in Streptomyces avermitilis.

The heat shock response (HSR) is a universal cellular response that promotes survival following temperature increase. In filamentous Streptomyces, which accounts for ∼70% of commercial antibiotic production, HSR is regulated by transcriptional repressors; in particular, the widespread MerR-family regulator HspR has been identified as a key repressor. However, functions of HspR in other biological processes are unknown. The present study demonstrates that HspR pleiotropically controls avermectin production, morphological development, and heat shock and H2O2 stress responses in the industrially important species Streptomyces avermitilis. HspR directly activated ave structural genes (aveA1 and aveA2) and H2O2 stress-related genes (katA1, catR, katA3, oxyR, ahpC, and ahpD), whereas it directly repressed heat shock genes (HSGs) (the dnaK1-grpE1-dnaJ1-hspR operon, clpB1p, clpB2p, and lonAp) and developmental genes (wblB, ssgY, and ftsH). HspR interacted with PhoP (response regulator of the widespread PhoPR two-component system) at dnaK1p to corepress the important dnaK1-grpE1-dnaJ1-hspR operon. PhoP exclusively repressed target HSGs (htpG, hsp18_1, and hsp18_2) different from those of HspR (clpB1p, clpB2p, and lonAp). A consensus HspR-binding site, 5'-TTGANBBNNHNNNDSTSHN-3', was identified within HspR target promoter regions, allowing prediction of the HspR regulon involved in broad cellular functions. Taken together, our findings demonstrate a key role of HspR in the coordination of a variety of important biological processes in Streptomyces species. IMPORTANCE Our findings are significant to clarify the molecular mechanisms underlying HspR function in Streptomyces antibiotic production, development, and H2O2 stress responses through direct control of its target genes associated with these biological processes. HspR homologs described to date function as transcriptional repressors but not as activators. The results of the present study demonstrate that HspR acts as a dual repressor/activator. PhoP cross talks with HspR at dnaK1p to coregulate the heat shock response (HSR), but it also has its own specific target heat shock genes (HSGs). The novel role of PhoP in the HSR further demonstrates the importance of this regulator in Streptomyces. Overexpression of hspR strongly enhanced avermectin production in Streptomyces avermitilis wild-type and industrial strains. These findings provide new insights into the regulatory roles and mechanisms of HspR and PhoP and facilitate methods for antibiotic overproduction in Streptomyces species.

Resistance-guided isolation and characterization of antibiotic-producing bacteria from river sediments.

BACKGROUND: To tackle the problem of antibiotic resistance, an extensive search for novel antibiotics is one of the top research priorities. Around 60% of the antibiotics used today were obtained from the genus Streptomyces. The river sediments of Bangladesh are still an unexplored source for antibiotic-producing bacteria (APB). This study aimed to isolate novel APB from Padma and Kapotakkho river sediments having the potential to produce antibacterial compounds with known scaffolds by manipulating their self-protection mechanisms. RESULTS: The antibiotic supplemented starch-casein-nitrate agar (SCNA) media were used to isolate antibiotic-resistant APB from the river sediments. The colonies having Streptomyces-like morphology were selectively purified and their antagonistic activity was screened against a range of test bacteria using the cross-streaking method. A notable decrease of the colony-forming units (CFUs) in the antibiotic supplemented SCNA plates compared to control plates (where added antibiotics were absent) was observed. A total of three azithromycin resistant (AZR) and nine meropenem resistant (MPR) isolates were purified and their antagonistic activity was investigated against a series of test bacteria including Shigella brodie, Escherichia coli, Pseudomonas sp., Proteus sp., Staphylococcus aureus, and Bacillus cereus. All the AZR isolates and all but two MPR isolates exhibited moderate to high broad-spectrum activity. Among the isolates, 16S rDNA sequencing of NAr5 and NAr6 were performed to identify them up to species level. The analyses of the sequences revealed that both belong to the genus Streptomyces. CONCLUSIONS: The results from these studies suggest that manipulation of the self-resistance property of APB is an easy and quick method to search for novel APB having the potential to produce potentially novel antibacterial compounds with known scaffolds.

Anti-Infective and Antiviral Activity of Valinomycin and Its Analogues from a Sea Cucumber-Associated Bacterium, Streptomyces sp. SV 21.

The manuscript investigated the isolation, characterization and anti-infective potential of valinomycin (3), streptodepsipeptide P11A (2), streptodepsipeptide P11B (1), and one novel valinomycin analogue, streptodepsipeptide SV21 (4), which were all produced by the Gram-positive strain Streptomycescavourensis SV 21. Although the exact molecular weight and major molecular fragments were recently reported for compound 4, its structure elucidation was not based on compound isolation and spectroscopic techniques. We successfully isolated and elucidated the structure based on the MS2 fragmentation pathways as well as 1H and 13C NMR spectra and found that the previously reported structure of compound 4 differs from our analysis. Our findings showed the importance of isolation and structure elucidation of bacterial compounds in the era of fast omics technologies. The here performed anti-infective assays showed moderate to potent activity against fungi, multi drug resistant (MDR) bacteria and infectivity of the Hepatitis C Virus (HCV). While compounds 2, 3 and 4 revealed potent antiviral activity, the observed minor cytotoxicity needs further investigation. Furthermore, the here performed anti-infective assays disclosed that the symmetry of the valinomycin molecule is most important for its bioactivity, a fact that has not been reported so far.

Discovery of Antibiofilm Activity of Elasnin against Marine Biofilms and Its Application in the Marine Antifouling Coatings.

Biofilms are surface-attached multicellular communities that play critical roles in inducing biofouling and biocorrosion in the marine environment. Given the serious economic losses and problems caused by biofouling and biocorrosion, effective biofilm control strategies are highly sought after. In a screening program of antibiofilm compounds against marine biofilms, we discovered the potent biofilm inhibitory activity of elasnin. Elasnin effectively inhibited the biofilm formation of seven strains of bacteria isolated from marine biofilms. With high productivity, elasnin-based coatings were prepared in an easy and cost-effective way, which exhibited great performance in inhibiting the formation of multi-species biofilms and the attachment of large biofouling organisms in the marine environment. The 16S amplicon analysis and anti-larvae assay revealed that elasnin could prevent biofouling by the indirect impact of changed microbial composition of biofilms and direct inhibitory effect on larval settlement with low toxic effects. These findings indicated the potential application of elasnin in biofilm and biofouling control in the marine environment.

Evolutionary stability of antibiotic protection in a defensive symbiosis.

The increasing resistance of human pathogens severely limits the efficacy of antibiotics in medicine, yet many animals, including solitary beewolf wasps, successfully engage in defensive alliances with antibiotic-producing bacteria for millions of years. Here, we report on the in situ production of 49 derivatives belonging to three antibiotic compound classes (45 piericidin derivatives, 3 streptochlorin derivatives, and nigericin) by the symbionts of 25 beewolf host species and subspecies, spanning 68 million years of evolution. Despite a high degree of qualitative stability in the antibiotic mixture, we found consistent quantitative differences between species and across geographic localities, presumably reflecting adaptations to combat local pathogen communities. Antimicrobial bioassays with the three main components and in silico predictions based on the structure and specificity in polyketide synthase domains of the piericidin biosynthesis gene cluster yield insights into the mechanistic basis and ecoevolutionary implications of producing a complex mixture of antimicrobial compounds in a natural setting.

Whole genome sequence of the multi-resistant plant growth-promoting bacteria Streptomyces sp. Z38 with potential application in agroindustry and bio-nanotechnology.

The genus Streptomyces is widely recognized for its biotechnological potential. Due to a need to improve crops, clean up the environment and produce novel antimicrobial molecules exploiting Streptomyces has become a priority. To further explore the biotechnological potential of these organisms we analyzed the genome of the strain Streptomyces sp. Z38 isolated from contaminated roots tissues. Our analysis not only confirmed the ability of the strain to produce plant growth promoting traits but also a range of mechanisms to cope with the toxic effect of heavy metals through genes involved in metal homeostasis and oxidative stress response. The production of silver nanoparticles indicated that Streptomyces sp. Z38 may find utility in Green, Grey and Red biotechnology.

New Scabimycins A-C Isolated from Streptomyces acidiscabies (Lu19992).

Peptide natural products displaying a wide range of biological activities have become important drug candidates over the years. Microorganisms have been a powerful source of such bioactive peptides, and Streptomyces have yielded many novel natural products thus far. In an effort to uncover such new, meaningful compounds, the metabolome of Streptomyces acidiscabies was analyzed thoroughly. Three new compounds, scabimycins A-C (1-3), were discovered, and their chemical structures were elucidated by NMR spectroscopy. The relative and absolute configurations were determined using ROESY NMR experiments and advanced Marfey's method.

Acylation and deacylation mechanism and kinetics of penicillin G reaction with Streptomyces R61 DD-peptidase.

Two quantum mechanical (QM)-cluster models are built for studying the acylation and deacylation mechanism and kinetics of Streptomyces R61 DD-peptidase with the penicillin G at atomic level detail. DD-peptidases are bacterial enzymes involved in the cross-linking of peptidoglycan to form the cell wall, necessary for bacterial survival. The cross-linking can be inhibited by antibiotic beta-lactam derivatives through acylation, preventing the acyl-enzyme complex from undergoing further deacylation. The deacylation step was predicted to be rate-limiting. Transition state and intermediate structures are found using density functional theory in this study, and thermodynamic and kinetic properties of the proposed mechanism are evaluated. The acyl-enzyme complex is found lying in a deep thermodynamic sink, and deacylation is indeed the severely rate-limiting step, leading to suicide inhibition of the peptidoglycan cross-linking. The usage of QM-cluster models is a promising technique to understand, improve, and design antibiotics to disrupt function of the Streptomyces R61 DD-peptidase.

Garcinoic Acids and a Benzophenone Derivative from the Seeds of Garcinia kola and Their Antibacterial Activities against Oral Bacterial Pathogenic Organisms.

In this study, three new garcinoic acid dimers, δ,δ-bigarcinoic acid (1), δ,δ-bi-O-garcinoic acid (2), and γ,δ-bi-O-garcinoic acid (3), and a new benzophenone derivative, (8E)-4-geranyl-3,5-dihydroxybenzophenone (4), as well as seven known compounds (5-11) were isolated from the seeds of Garcinia kola. The structures of the new compounds were elucidated using MALDI-TOF-MS and spectroscopic data, including 1D and 2D NMR and electronic circular dichroism spectra. All of the isolated compounds were evaluated for their antimicrobial activity against two oral pathogens, Porphyromonas gingivalis and Streptococcus sobrinus. Among them, 4 and δ-garcinoic acid (6) exhibited antimicrobial activity against both of these microorganisms (MICs of 31.3-62.5 µM for P. gingivalis and 15.6-31.3 µM for S. sobrinus). These results indicate that some chemical constituents in G. kola seeds have potential application in the prevention of oral diseases.

Activation of paulomycin production by exogenous γ-butyrolactone signaling molecules in Streptomyces albidoflavus J1074.

The interspecies communication roles of γ-butyrolactones (GBLs) have been described for a long time but are still poorly understood. Herein, we analyzed more than 1000 Streptomyces strains and noticed a big quantitative gap between the strains with GBL biosynthetic genes and the strains with GBL receptor genes, which implies the wide-spread of GBLs as interspecies signals in Streptomyces and their great potential in the activation of silent natural product gene clusters. Streptomyces albidoflavus J1074, which has one GBL receptor gene but no GBL biosynthetic gene, was chosen as a target to study the possible interspecies communication roles of GBLs. At first, the GBL biosynthetic genes from Streptomyces coelicolor M145 were expressed in S. albidoflavus J1074, which enabled the S. albidoflavus strains to synthesize Streptomyces coelicolor butanolides (SCBs) and activated the production of paulomycins. Further studies showed that this activation process requires the participation of the GBL receptor gene XNR_4681. The results suggest that the expression of exogenous GBL biosynthetic genes can modulate the metabolisms of GBL non-producing strains, and this regulation role might be meaningful for silent gene cluster activation in Streptomyces. At final, we synthesized racemic-SCB2 and tried to simplify the activation process by adding SCB2 directly to S. albidoflavus J1074, which unfortunately failed to induce paulomycin production.

Genome mining and homologous comparison strategy for digging exporters contributing self-resistance in natamycin-producing Streptomyces strains.

As antibiotics are always toxic to the antibiotic-producing strains themselves, most Streptomyces strains have evolved several self-resistance mechanisms, among which the antibiotic efflux system is understood best and is commonly found. Among the efflux systems, the ATP-binding cassette (ABC) transporter superfamily and the major facilitator superfamily (MFS) are two important transporter families. In this work, the ABC transporters and the MFS transporters from the four reported natamycin-producing Streptomyces strains have been investigated in order to clarify whether these Streptomyces strains share similar efflux strategies for natamycin metabolism. Fifty-one groups of homologous exporter genes were identified as shared by four strains. Differential transcriptional analysis between the natamycin-producing strain Streptomyces chattanoogensis L10 and its ΔscnS0 mutant, which produces no natamycin, reveals that the expression levels of 25 of the above groups of genes were observably changed. The production of natamycin declined over 30% after solely knocking out several of these 25 groups of genes in S. chattanoogensis L10. This indicates that these transporters participate in the efflux of molecules related to natamycin biosynthesis. Our study is the first to demonstrate that the exporters participating in a particular antibiotic metabolism can be excavated and identified quickly by the strategy of genome mining and homologous comparison in the antibiotic-producing strains, leading to deeper understanding of the complex self-resistance mechanisms in Streptomycetes.

Fungal elicitor-induced transcriptional changes of genes related to branched-chain amino acid metabolism in Streptomyces natalensis HW-2.

Natamycin is a polyene macrolide antibiotic and widely used as a natural food preservative. Fungal elicitor had positive effects on the natamycin biosynthesis in Streptomyces natalensis HW-2. However, the global gene expression in response to fungal elicitor is not still reported. In the study, RNA-Seq was used to check the change of transcriptome by fungal elicitor in S. natalensis HW-2. The results showed that there were 1265 differential expression genes (DEGs) at 40 h and 2196 DEGs at 80 h. Most of the genes involved in natamycin biosynthesis were upregulated. KEGG pathway analysis showed that fungal elicitor had strong effects on the transcriptional levels of genes related to branch-chained amino acid (BCAA) metabolism. There were 23 upregulated or downregulated DEGs involved in BCAA biosynthesis and degradation at 40 h and 80 h. To confirm whether the improvement of BCAA biosynthesis could produce more natamycin, metabolic engineering was used to homologously overexpress the gene ilvH which encoded the regulatory subunit of acetolactate synthase (ALS) in S. natalensis. The results showed that overexpression of ilvH in S. natalensis HW-2 increased natamycin production to 1.25 g/L in the flask, which was a 32% increase compared with that of the parent strain. Real-time quantitative PCR analysis showed that the transcriptional level of ilvH in mutant strain S. natalensis ZS101 was significantly increased. Acetyl-CoA content was also raised. The results suggested that the fungal elicitor enhanced natamycin biosynthesis by improving precursor supply via BCAA metabolism. This study will open a new avenue for enhancing natamycin production by metabolic engineering and adding fungal elicitor. KEY POINTS: • The fungal elicitor had strong effects on the transcriptional levels of genes related to branch-chained amino acid metabolism by RNA-Seq. • The homologous overexpression of gene ilvH increased natamycin production by 32% and acetyl-CoA content was raised in mutant strain S. natalensis ZS101.

Intracellular Proteomic Analysis of Streptomyces sp. MC1 When Exposed to Cr(VI) by Gel-Based and Gel-Free Methods.

The actinobacterium Streptomyces sp. MC1 has previously shown the capacity to resist and remove Cr(VI) from liquid culture media. The aim of this work is to analyze the differential expression pattern of intracellular proteins when Streptomyces sp. MC1 is exposed to Cr(VI) in order to explain the molecular mechanisms of resistance that this microorganism possesses. For this purpose, 2D-PAGE and shotgun proteomic analyses (2D-nanoUPLC-ESI-MS/MS) were applied. The presence of Cr(VI) induced the expression of proteins involved in molecular biosynthesis and energy generation, chaperones with a key role in the repair of misfolded proteins and stress response, transcription proteins, proteins of importance in the DNA supercoiling, repair and replication, and dehydrogenases involved in oxidation-reduction processes. These dehydrogenases can be associated with the reduction of Cr(VI) to Cr(III). The results of this study show that proteins from the groups mentioned before are important to face the stress caused by the Cr(VI) presence and help the microorganism to counteract the toxicity of the metal. The use of two proteomic approaches resulted in a larger number of peptides identified, which is also transduced in a significant number of protein ID. This decreased the potential complexity of the sample because of the protein dynamic range, as well as increased the recovery of peptides from the gel after digestion.

Three putative DNA replication/repair elements encoding genes confer self-resistance to distamycin in Streptomyces netropsis.

Distamycin (DST) is a well-characterized DNA minor groove binder with antivirus activity and antitumor potency. Two separate gene clusters (a 28-kb cluster and a 7-kb cluster) have recently been identified to coordinately encode the biosynthetic machinery of DST in Streptomyces netropsis. Here we report a gene cassette, which is linked to the aforementioned smaller dst gene cluster and plays an important role in the self-resistance to DST in S. netropsis. This cassette consists of three uncharacterized genes that might be implicated in DNA replication/repair. Knockout of the cassette led to the decrease in the production of DST, while heterologous expression of part of the cassette in S. lividans made it become resistant to both DST and mitomycin C, another DNA-binding agent. More interestingly, homologs of these three genes were found in genomes of other actinomyces that produce DNA-binding antibiotics, suggesting that a novel common mechanism in addition to pumping may enable these strains to resist the cytotoxic metabolites they produced.

Biosynthesis of the pyrrolidine protein synthesis inhibitor anisomycin involves novel gene ensemble and cryptic biosynthetic steps.

The protein synthesis inhibitor anisomycin features a unique benzylpyrrolidine system and exhibits diverse biological and pharmacologic activities. Its biosynthetic origin has remained obscure for more than 60 y, however. Here we report the identification of the biosynthetic gene cluster (BGC) of anisomycin in Streptomyces hygrospinosus var. beijingensis by a bioactivity-guided high-throughput screening method. Using a combination of bioinformatic analysis, reverse genetics, chemical analysis, and in vitro biochemical assays, we have identified a core four-gene ensemble responsible for the synthesis of the pyrrolidine system in anisomycin: aniQ, encoding a aminotransferase that catalyzes an initial deamination and a later reamination steps; aniP, encoding a transketolase implicated to bring together an glycolysis intermediate with 4-hydroxyphenylpyruvic acid to form the anisomycin molecular backbone; aniO, encoding a glycosyltransferase that catalyzes a cryptic glycosylation crucial for downstream enzyme processing; and aniN, encoding a bifunctional dehydrogenase that mediates multistep pyrrolidine formation. The results reveal a BGC for pyrrolidine alkaloid biosynthesis that is distinct from known bacterial alkaloid pathways, and provide the signature sequences that will facilitate the discovery of BGCs encoding novel pyrrolidine alkaloids in bacterial genomes. The biosynthetic insights from this study further set the foundation for biosynthetic engineering of pyrrolidine antibiotics.