A comparative metabologenomic approach reveals mechanistic insights into Streptomyces antibiotic crypticity

Streptomyces genomes harbor numerous, biosynthetic gene clusters (BGCs) encoding for drug-like compounds. While some of these BGCs readily yield expected products, many do not. Biosynthetic crypticity represents a significant hurdle to drug discovery, and the biological mechanisms that underpin it remain poorly understood. Polycyclic tetramate macrolactam (PTM) antibiotic production is widespread within the Streptomyces genus, and examples of active and cryptic PTM BGCs are known. To reveal further insights into the causes of biosynthetic crypticity, we employed a PTM-targeted comparative metabologenomics approach to analyze a panel of S. griseus clade strains that included both poor and robust PTM producers. By comparing the genomes and PTM production profiles of these strains, we systematically mapped the PTM promoter architecture within the group, revealed that these promoters are directly activated via the global regulator AdpA, and discovered that small promoter insertion–deletion lesions (indels) differentiate weaker PTM producers from stronger ones. We also revealed an unexpected link between robust PTM expression and griseorhodin pigment coproduction, with weaker S. griseus–clade PTM producers being unable to produce the latter compound. This study highlights promoter indels and biosynthetic interactions as important, genetically encoded factors that impact BGC outputs, providing mechanistic insights that will undoubtedly extend to other Streptomyces BGCs. We highlight comparative metabologenomics as a powerful approach to expose genomic features that differentiate strong, antibiotic producers from weaker ones. This should prove useful for rational discovery efforts and is orthogonal to current engineering and molecular signaling approaches now standard in the field.

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MtrA is an essential regulator that coordinates antibiotic production and sporulation in Streptomyces species

10.1101/090399 ◽

2016 ◽

Cited By ~ 3

Author(s):

Nicolle F. Som ◽

Daniel Heine ◽

John T. Munnoch ◽

Neil A. Holmes ◽

Felicity Knowles ◽

...

Keyword(s):

Secondary Metabolism ◽

Response Regulator ◽

Antibiotic Production ◽

Gene Clusters ◽

Antibiotic Biosynthesis ◽

Biosynthetic Gene ◽

Global Regulator ◽

Major Focus ◽

Biosynthetic Gene Clusters ◽

Streptomyces Species

AbstractStreptomyces bacteria make numerous secondary metabolites, including half of all known antibiotics. Understanding the global regulation of secondary metabolism is important because most Streptomyces natural products are not made under laboratory conditions and unlocking ‘cryptic’ biosynthetic gene clusters (BGCs) is a major focus for natural product discovery. Production is coordinated with sporulation but the regulators that coordinate development with antibiotic biosynthesis are largely unknown. Here we characterise a highly conserved actinobacterial response regulator called MtrA in antibiotic-producing Streptomyces species. We show that MtrA is an essential global regulator of secondary metabolism that directly activates antibiotic production in in S. coelicolor and S. venezuelae. MtrA also controls key developmental genes required for DNA replication and cell division and we propose that MtrA is the missing link that coordinates secondary metabolism with development in Streptomyces species.

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Identification of a New Antimicrobial, Desertomycin H, Utilizing a Modified Crowded Plate Technique

Marine Drugs ◽

10.3390/md19080424 ◽

2021 ◽

Vol 19 (8) ◽

pp. 424

Author(s):

Osama G. Mohamed ◽

Sadaf Dorandish ◽

Rebecca Lindow ◽

Megan Steltz ◽

Ifrah Shoukat ◽

...

Keyword(s):

Antibiotic Production ◽

Gene Clusters ◽

Multidrug Resistant ◽

Microbial Interactions ◽

Mass Spectrometry Data ◽

Metagenomic Data ◽

Resistant Bacteria ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Plate Technique

The antibiotic-resistant bacteria-associated infections are a major global healthcare threat. New classes of antimicrobial compounds are urgently needed as the frequency of infections caused by multidrug-resistant microbes continues to rise. Recent metagenomic data have demonstrated that there is still biosynthetic potential encoded in but transcriptionally silent in cultivatable bacterial genomes. However, the culture conditions required to identify and express silent biosynthetic gene clusters that yield natural products with antimicrobial activity are largely unknown. Here, we describe a new antibiotic discovery scheme, dubbed the modified crowded plate technique (mCPT), that utilizes complex microbial interactions to elicit antimicrobial production from otherwise silent biosynthetic gene clusters. Using the mCPT as part of the antibiotic crowdsourcing educational program Tiny Earth®, we isolated over 1400 antibiotic-producing microbes, including 62, showing activity against multidrug-resistant pathogens. The natural product extracts generated from six microbial isolates showed potent activity against vancomycin-intermediate resistant Staphylococcus aureus. We utilized a targeted approach that coupled mass spectrometry data with bioactivity, yielding a new macrolactone class of metabolite, desertomycin H. In this study, we successfully demonstrate a concept that significantly increased our ability to quickly and efficiently identify microbes capable of the silent antibiotic production.

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Phytoplankton trigger the production of cryptic metabolites in the marine actinobacteria Salinispora tropica

10.1101/2020.05.18.103358 ◽

2020 ◽

Author(s):

Audam Chhun ◽

Despoina Sousoni ◽

Maria del Mar Aguiló-Ferretjans ◽

Lijiang Song ◽

Christophe Corre ◽

...

Keyword(s):

Natural Products ◽

Secondary Metabolites ◽

Antibiotic Production ◽

Antimicrobial Effect ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Marine Actinobacteria ◽

Salinispora Tropica ◽

Eukaryotic Phytoplankton

AbstractBacteria from the Actinomycete family are a remarkable source of natural products with pharmaceutical potential. The discovery of novel molecules from these organisms is, however, hindered because most of the biosynthetic gene clusters (BGCs) encoding these secondary metabolites are cryptic or silent and are referred to as orphan BGCs. While co-culture has proven to be a promising approach to unlock the biosynthetic potential of many microorganisms by activating the expression of these orphan BGCs, it still remains an underexplored technique. The marine actinobacteria Salinispora tropica, for instance, produces valuable compounds such as the anti-cancer molecule salinosporamide A but half of its putative BGCs are still orphan. Although previous studies have looked into using marine heterotrophs to induce orphan BGCs in Salinispora, the potential impact of co-culturing marine phototrophs with Salinispora has yet to be investigated. Following the observation of clear antimicrobial phenotype of the actinobacterium on a range of phytoplanktonic organisms, we here report the discovery of novel cryptic secondary metabolites produced by S. tropica in response to its co-culture with photosynthetic primary producers. An approach combining metabolomics and proteomics revealed that the photosynthate released by phytoplankton influences the biosynthetic capacities of S. tropica with both production of new molecules and the activation of orphan BGCs. Our work pioneers the use of phototrophs as a promising strategy to accelerate the discovery of novel natural products from actinobacteria.ImportanceThe alarming increase of antimicrobial resistance has generated an enormous interest in the discovery of novel active compounds. The isolation of new microbes to untap novel natural products is currently hampered because most biosynthetic gene clusters (BGC) encoded by these microorganisms are not expressed under standard laboratory conditions, i.e. mono-cultures. Here we show that co-culturing can be an easy way for triggering silent BGC. By combining state-of-the-art metabolomics and high-throughput proteomics, we characterized the activation of cryptic metabolites and silent biosynthetic gene clusters in the marine actinobacteria Salinispora tropica by the presence of phytoplankton photosynthate. We further suggest a mechanistic understanding of the antimicrobial effect this actinobacterium has on a broad range of prokaryotic and eukaryotic phytoplankton species and reveal a promising candidate for antibiotic production.

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Competition sensing alters antibiotic production in Streptomyces

10.1101/2020.01.24.918557 ◽

2020 ◽

Cited By ~ 2

Author(s):

Sanne Westhoff ◽

Alexander Kloosterman ◽

Stephan F. A. van Hoesel ◽

Gilles P. van Wezel ◽

Daniel E. Rozen

Keyword(s):

Genetic Relatedness ◽

Cell Damage ◽

Antibiotic Production ◽

Interference Competition ◽

Resource Limitation ◽

Gene Clusters ◽

Regulatory Control ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

The Social

ABSTRACTOne of the most important ways that bacteria compete for resources and space is by producing antibiotics that inhibit competitors. Because antibiotic production is costly, the biosynthetic gene clusters coordinating their synthesis are under strict regulatory control and often require “elicitors” to induce expression, including cues from competing strains. Although these cues are common, they are not produced by all competitors and so the phenotypes causing induction remain unknown. By studying interactions between 24 antibiotic-producing streptomycetes we show that inhibition between competitors is common and occurs more frequently if strains are closely related. Next, we show that antibiotic production is more likely to be induced by cues from strains that are closely related or that share secondary metabolite biosynthetic gene clusters (BGCs). Unexpectedly, antibiotic production is less likely to be induced by competitors that inhibit the growth of a focal strain, indicating that cell damage is not a general cue for induction. In addition to induction, antibiotic production often decreased in the presence of a competitor, although this response was not associated with genetic relatedness or overlap in BGCs. Finally, we show that resource limitation increases the chance that antibiotic production declines during competion. Our results reveal the importance of social cues and resource availability in the dynamics of interference competition in streptomycetes.SIGNIFICANCE STATEMENTBacteria secrete antibiotics to inhibit their competitors, but the presence of competitors can determine whether these toxins are produced. Here, we study the role of the competitive and resource environment on antibiotic production in Streptomyces, bacteria renowned for their production of antibiotics. We show that Streptomyces are more likely to produce antibiotics when grown with closely related competitors or that share biosynthetic pathways for secondary metabolites, but not when they are threatened by competitor’s toxins, in contrast to predictions of the competition sensing hypothesis. Streptomyces also often reduce their output of antibiotics when grown with competitors, especially under nutrient limitation. Our findings highlight that interactions between the social and resource environments strongly regulate antibiotic production in these medicinally important bacteria.

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The Antibiotic Andrimid Produced by Vibrio coralliilyticus Increases Expression of Biosynthetic Gene Clusters and Antibiotic Production in Photobacterium galatheae

Frontiers in Microbiology ◽

10.3389/fmicb.2020.622055 ◽

2020 ◽

Vol 11 ◽

Author(s):

Yannick Buijs ◽

Thomas Isbrandt ◽

Sheng-Da Zhang ◽

Thomas Ostenfeld Larsen ◽

Lone Gram

Keyword(s):

Stress Response ◽

Antibiotic Production ◽

Gene Clusters ◽

Multidrug Resistant ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Gene Encoding ◽

Chemical Measurements ◽

Low Concentrations ◽

Vibrio Coralliilyticus

The development and spread of multidrug resistant pathogens have reinforced the urgency to find novel natural products with antibiotic activity. In bacteria, orphan biosynthetic gene clusters (BGCs) far outnumber the BGCs for which chemistry is known, possibly because they are transcriptionally silent under laboratory conditions. A strategy to trigger the production of this biosynthetic potential is to challenge the microorganism with low concentrations of antibiotics, and by using a Burkholderia genetic reporter strain (Seyedsayamdost, Proc Natl Acad Sci 111:7266–7271), we found BGC unsilencing activity for the antimicrobial andrimid, produced by the marine bacterium Vibrio coralliilyticus. Next, we challenged another marine Vibrionaceae, Photobacterium galatheae, carrier of seven orphan BGCs with sub-inhibitory concentrations of andrimid. A combined approach of transcriptional and chemical measurements of andrimid-treated P. galatheae cultures revealed a 10-fold upregulation of an orphan BGC and, amongst others, a 1.6–2.2-fold upregulation of the gene encoding the core enzyme for biosynthesis of holomycin. Also, addition of andrimid caused an increase, based on UV-Vis peak area, of 4-fold in production of the antibiotic holomycin. Transcriptional measurements of stress response related genes in P. galatheae showed a co-occurrence of increased transcript levels of rpoS (general stress response) and andrimid induced holomycin overproduction, while in trimethoprim treated cultures attenuation of holomycin production coincided with a transcriptional increase of recA (SOS stress response). This study shows that using antimicrobial compounds as activators of secondary metabolism can be a useful strategy in eliciting biosynthetic gene clusters and facilitate natural product discovery. Potentially, such interactions could also have ecological relevant implications.

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Microbiological and molecular insights on rare Actinobacteria harboring bioactive prospective

Bulletin of the National Research Centre ◽

10.1186/s42269-019-0266-8 ◽

2020 ◽

Vol 44 (1) ◽

Author(s):

Dina H. Amin ◽

Nagwa A. Abdallah ◽

Assem Abolmaaty ◽

Sahar Tolba ◽

Elizabeth M. H. Wellington

Keyword(s):

Bioinformatics Analysis ◽

Genetic Manipulation ◽

Antibiotic Production ◽

Gene Clusters ◽

Bioactive Molecules ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Pcr Screening ◽

Molecular Approaches ◽

Shed Light

Abstract Background Actinobacteria is as a group of advanced filamentous bacteria. Rare Actinobacteria are of special interest as they are rarely isolated from the environments. They are a major source of important bioactive compounds. Determining the proper strategy for the identification of Actinobacteria harboring biosynthetic gene clusters and producing bioactive molecules is a challenging platform. Methodology In this review, we discuss a consequence of microbiological and molecular methods for the identification of rare Actinobacteria. In addition to that, we shed light on rare Actinobacteria’s significance in antibiotic production. We also clarified molecular approaches for the manipulation of novel biosynthetic gene clusters via PCR screening, fosmid libraries, and Illumina whole-genome sequencing in combination with bioinformatics analysis. Conclusion Perceptions of the conventional and molecular identification of Actinobacteria were conducted. This will open the door for the genetic manipulation of novel antibiotic gene clusters in heterologous hosts. Also, these conclusions will lead to constructing new bioactive molecules via genetically engineering biosynthetic pathways.

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Competition Sensing Changes Antibiotic Production in Streptomyces

mBio ◽

10.1128/mbio.02729-20 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Sanne Westhoff ◽

Alexander M. Kloosterman ◽

Stephan F. A. van Hoesel ◽

Gilles P. van Wezel ◽

Daniel E. Rozen

Keyword(s):

Genetic Relatedness ◽

Cell Damage ◽

Antibiotic Production ◽

Interference Competition ◽

Resource Limitation ◽

Gene Clusters ◽

Regulatory Control ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

The Social

ABSTRACT One of the most important ways that bacteria compete for resources and space is by producing antibiotics that inhibit competitors. Because antibiotic production is costly, the biosynthetic gene clusters coordinating their synthesis are under strict regulatory control and often require “elicitors” to induce expression, including cues from competing strains. Although these cues are common, they are not produced by all competitors, and so the phenotypes causing induction remain unknown. By studying interactions between 24 antibiotic-producing strains of streptomycetes, we show that strains commonly inhibit each other’s growth and that this occurs more frequently if strains are closely related. Next, we show that antibiotic production is more likely to be induced by cues from strains that are closely related or that share secondary metabolite biosynthetic gene clusters (BGCs). Unexpectedly, antibiotic production is less likely to be induced by competitors that inhibit the growth of a focal strain, indicating that cell damage is not a general cue for induction. In addition to induction, antibiotic production often decreases in the presence of a competitor, although this response was not associated with genetic relatedness or overlap in BGCs. Finally, we show that resource limitation increases the chance that antibiotic production declines during competition. Our results reveal the importance of social cues and resource availability in the dynamics of interference competition in streptomycetes. IMPORTANCE Bacteria secrete antibiotics to inhibit their competitors, but the presence of competitors can determine whether these toxins are produced. Here, we study the role of the competitive and resource environment on antibiotic production in Streptomyces, bacteria renowned for their production of antibiotics. We show that Streptomyces cells are more likely to produce antibiotics when grown with competitors that are closely related or that share biosynthetic pathways for secondary metabolites, but not when they are threatened by competitor’s toxins, in contrast to predictions of the competition sensing hypothesis. Streptomyces cells also often reduce their output of antibiotics when grown with competitors, especially under nutrient limitation. Our findings highlight that interactions between the social and resource environments strongly regulate antibiotic production in these medicinally important bacteria.

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Understanding and manipulating antibiotic production in actinomycetes

Biochemical Society Transactions ◽

10.1042/bst20130214 ◽

2013 ◽

Vol 41 (6) ◽

pp. 1355-1364 ◽

Cited By ~ 29

Author(s):

Mervyn J. Bibb

Keyword(s):

Natural Product ◽

Biological Activities ◽

Antibiotic Production ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Natural Product Biosynthesis ◽

Genomic Technologies ◽

Wide Range ◽

Recent Developments

Actinomycetes are prolific producers of natural products with a wide range of biological activities. Many of the compounds that they make (and derivatives thereof) are used extensively in medicine, most notably as clinically important antibiotics, and in agriculture. Moreover, these organisms remain a source of novel and potentially useful molecules, but maximizing their biosynthetic potential requires a better understanding of natural product biosynthesis. Recent developments in genome sequencing have greatly facilitated the identification of natural product biosynthetic gene clusters. In the present article, I summarize the recent contributions of our laboratory in applying genomic technologies to better understand and manipulate natural product biosynthesis in a range of different actinomycetes.

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Heterologous Expression of Novobiocin and Clorobiocin Biosynthetic Gene Clusters

Applied and Environmental Microbiology ◽

10.1128/aem.71.5.2452-2459.2005 ◽

2005 ◽

Vol 71 (5) ◽

pp. 2452-2459 ◽

Cited By ~ 76

Author(s):

Alessandra S. Eustáquio ◽

Bertolt Gust ◽

Ute Galm ◽

Shu-Ming Li ◽

Keith F. Chater ◽

...

Keyword(s):

Heterologous Expression ◽

Antibiotic Production ◽

Streptomyces Coelicolor ◽

Attachment Site ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Gene Deletions ◽

Or Gene ◽

Genetically Manipulated

ABSTRACT A method was developed for the heterologous expression of biosynthetic gene clusters in different Streptomyces strains and for the modification of these clusters by single or multiple gene replacements or gene deletions with unprecedented speed and versatility. λ-Red-mediated homologous recombination was used for genetic modification of the gene clusters, and the attachment site and integrase of phage φC31 were employed for the integration of these clusters into the heterologous hosts. This method was used to express the gene clusters of the aminocoumarin antibiotics novobiocin and clorobiocin in the well-studied strains Streptomyces coelicolor and Streptomyces lividans, which, in contrast to the natural producers, can be easily genetically manipulated. S. coelicolor M512 derivatives produced the respective antibiotic in yields comparable to those of natural producer strains, whereas S. lividans TK24 derivatives were at least five times less productive. This method could also be used to carry out functional investigations. Shortening of the cosmids' inserts showed which genes are essential for antibiotic production.

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Adrenaline-mediated activation of antibiotic production in Streptomyces highlights catechol as elicitor of specialized metabolism

10.21203/rs.3.rs-715051/v1 ◽

2021 ◽

Author(s):

Gilles van Wezel ◽

Doris van Bergeijk ◽

Somayah Elsayed ◽

Chao Du ◽

Isabel Nunez Santiago ◽

...

Keyword(s):

Antibiotic Production ◽

Gene Clusters ◽

Bioactive Molecules ◽

Stress Hormone ◽

Biosynthetic Gene Clusters ◽

Mass Spectral ◽

Catechol 1,2 Dioxygenase ◽

Human Stress ◽

First Time ◽

Catechol Moiety

Abstract Actinobacteria are a rich source of bioactive molecules, and genome sequencing has shown that the vast majority of their biosynthetic potential has yet to be explored. However, many of their biosynthetic gene clusters (BGCs) are poorly expressed in the laboratory, which prevents discovery of their cognate natural products. To exploit their full biosynthetic potential, better understanding of the signals that promote the expression of BGCs is needed. Here, we show that the human stress hormone epinephrine (adrenaline) elicits antibiotic production by Actinobacteria. Catechol was established as the likely eliciting moiety, since similar responses were seen for catechol and for the catechol-containing molecules dopamine and catechin but not for related molecules. Exploration of the catechol-responsive strain Streptomyces sp. MBT84 using mass spectral networking revealed elicitation of a BGC that produces the angucycline glycosides aquayamycin, urdamycinone B and galtamycin C. Heterologous expression of the catechol-cleaving enzymes catechol 1,2-dioxygenase or catechol 2,3 dioxygenase counteracted the eliciting effect of catechol. Thus, for the first time we show the activation of natural product biosynthesis by a human hormone, leading to the identification of the ubiquitous catechol moiety as elicitor of BGCs for siderophores and antibiotics.

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