Unique physiological and genetic features of ofloxacin-resistant Streptomyces mutants

New antimicrobial agents are urgently needed to combat the emergence and spread of multidrug-resistant bacteria. Activating the cryptic biosynthetic gene clusters for actinomycete secondary metabolites can provide essential clues for research into new antimicrobial agents. An effective method for this purpose is based on drug resistance selection. This report describes interesting results for drug resistance selection using antibiotics that target DNA replication can effectively potentiate secondary metabolite production by actinomycetes. Ofloxacin-resistant mutants were isolated from five different streptomycetes. Ofloxacin is an antibiotic that binds to DNA complexes and type II topoisomerase, causing double-stranded breaks in bacterial chromosomes. Physiological and genetic characterization of the mutants revealed that the development of ofloxacin resistance in streptomycetes leads to the emergence of various types of secondary metabolite-overproducing strains. In Streptomyces coelicolor A3(2), ofloxacin-resistant mutants that overproduced actinorhodin, undecylprodigiosin, or carotenoid were identified. Also, an ofloxacin-resistant mutant that overproduces methylenomycin A, whose biosynthetic gene cluster is located on the endogenous plasmid, SCP1, was isolated. These observations indicate that ofloxacin resistance might activate biosynthetic genes on both chromosomes and on endogenous plasmids. We also identified the mutations that are probably involved in the phenotype of ofloxacin resistance and secondary metabolite overproduction in S. coelicolor A3(2). Furthermore, we observed an interesting phenomenon in which several ofloxacin-resistant mutants overproduced antibiotics in the presence of ofloxacin. Based on these results, we present the unique physiological and genetic characteristics of ofloxacin-resistant Streptomyces mutants and discuss the importance and potential development of the new findings. IMPORTANCE The abuse or overuse of antibacterial agents for therapy and animal husbandry has caused an increased population of antimicrobial-resistant bacteria in the environment. Consequently, there are now fewer effective antimicrobials available. Due to the depleted antibiotic pipeline, pandemic outbreaks caused by antimicrobial-resistant bacteria are deeply concerned, and the development of new antibiotics is now an urgent issue. Promising sources of antimicrobial agents include cryptic biosynthetic gene clusters for secondary metabolites in streptomycetes and rare actinomycetes. This study’s significance is an unprecedented activation method to accelerate drug discovery research on a global scale. The technique developed in this study could allow for simultaneous drug discovery in different countries, maximizing the world’s microbial resources.

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Phylogenetic Distribution of Secondary Metabolites in the Bacillus subtilis Species Complex

mSystems ◽

10.1128/msystems.00057-21 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

Kat Steinke ◽

Omkar S. Mohite ◽

Tilmann Weber ◽

Ákos T. Kovács

Keyword(s):

Bacillus Subtilis ◽

Secondary Metabolites ◽

Secondary Metabolite ◽

Species Complex ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Content Type ◽

Frameshift Mutations ◽

Metabolite Production

ABSTRACT Microbes produce a plethora of secondary (or specialized) metabolites that, although not essential for primary metabolism, benefit them to survive in the environment, communicate, and influence cell differentiation. Biosynthetic gene clusters (BGCs), responsible for the production of these secondary metabolites, are readily identifiable on bacterial genome sequences. Understanding the phylogeny and distribution of BGCs helps us to predict the natural product synthesis ability of new isolates. Here, we examined 310 genomes from the Bacillus subtilis group, determined the inter- and intraspecies patterns of absence/presence for all BGCs, and assigned them to defined gene cluster families (GCFs). This allowed us to establish patterns in the distribution of both known and unknown products. Further, we analyzed variations in the BGC structures of particular families encoding natural products, such as plipastatin, fengycin, iturin, mycosubtilin, and bacillomycin. Our detailed analysis revealed multiple GCFs that are species or clade specific and a few others that are scattered within or between species, which will guide exploration of the chemodiversity within the B. subtilis group. Surprisingly, we discovered that partial deletion of BGCs and frameshift mutations in selected biosynthetic genes are conserved within phylogenetically related isolates, although isolated from around the globe. Our results highlight the importance of detailed genomic analysis of BGCs and the remarkable phylogenetically conserved erosion of secondary metabolite biosynthetic potential in the B. subtilis group. IMPORTANCE Members of the B. subtilis species complex are commonly recognized producers of secondary metabolites, among those, the production of antifungals, which makes them promising biocontrol strains. While there are studies examining the distribution of well-known secondary metabolites in Bacilli, intraspecies clade-specific distribution has not been systematically reported for the B. subtilis group. Here, we report the complete biosynthetic potential within the B. subtilis group to explore the distribution of the biosynthetic gene clusters and to reveal an exhaustive phylogenetic conservation of secondary metabolite production within Bacillus that supports the chemodiversity within this species complex. We identify that certain gene clusters acquired deletions of genes and particular frameshift mutations, rendering them inactive for secondary metabolite biosynthesis, a conserved genetic trait within phylogenetically conserved clades of certain species. The overview guides the assignment of the secondary metabolite production potential of newly isolated Bacillus strains based on genome sequence and phylogenetic relatedness.

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Chromatin-dependent regulation of secondary metabolite biosynthesis in fungi: is the picture complete?

FEMS Microbiology Reviews ◽

10.1093/femsre/fuz018 ◽

2019 ◽

Cited By ~ 4

Author(s):

Jérôme Collemare ◽

Michael F Seidl

Keyword(s):

Secondary Metabolites ◽

Secondary Metabolite ◽

Biological Activities ◽

Gene Clusters ◽

Research Field ◽

Chromatin Modifications ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Post Translational Modifications ◽

Fungal Secondary Metabolites

ABSTRACTFungal secondary metabolites are small molecules that exhibit diverse biological activities exploited in medicine, industry and agriculture. Their biosynthesis is governed by co-expressed genes that often co-localize in gene clusters. Most of these secondary metabolite gene clusters are inactive under laboratory conditions, which is due to a tight transcriptional regulation. Modifications of chromatin, the complex of DNA and histone proteins influencing DNA accessibility, play an important role in this regulation. However, tinkering with well-characterised chemical and genetic modifications that affect chromatin alters the expression of only few biosynthetic gene clusters, and thus the regulation of the vast majority of biosynthetic pathways remains enigmatic. In the past, attempts to activate silent gene clusters in fungi mainly focused on histone acetylation and methylation, while in other eukaryotes many other post-translational modifications are involved in transcription regulation. Thus, how chromatin regulates the expression of gene clusters remains a largely unexplored research field. In this review, we argue that focusing on only few well-characterised chromatin modifications is significantly hampering our understanding of the chromatin-based regulation of biosynthetic gene clusters. Research on underexplored chromatin modifications and on the interplay between different modifications is timely to fully explore the largely untapped reservoir of fungal secondary metabolites.

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Comprehensive Analyses of Cytochrome P450 Monooxygenases and Secondary Metabolite Biosynthetic Gene Clusters in Cyanobacteria

International Journal of Molecular Sciences ◽

10.3390/ijms21020656 ◽

2020 ◽

Vol 21 (2) ◽

pp. 656 ◽

Cited By ~ 4

Author(s):

Makhosazana Jabulile Khumalo ◽

Nomfundo Nzuza ◽

Tiara Padayachee ◽

Wanping Chen ◽

Jae-Hyuk Yu ◽

...

Keyword(s):

Cytochrome P450 ◽

Secondary Metabolites ◽

Secondary Metabolite ◽

Gene Clusters ◽

Cytochrome P450 Monooxygenases ◽

Biosynthetic Gene ◽

Mycobacterial Species ◽

Biosynthetic Gene Clusters ◽

P450 Monooxygenases ◽

Cyanobacterial Species

The prokaryotic phylum Cyanobacteria are some of the oldest known photosynthetic organisms responsible for the oxygenation of the earth. Cyanobacterial species have been recognised as a prosperous source of bioactive secondary metabolites with antibacterial, antiviral, antifungal and/or anticancer activities. Cytochrome P450 monooxygenases (CYPs/P450s) contribute to the production and diversity of various secondary metabolites. To better understand the metabolic potential of cyanobacterial species, we have carried out comprehensive analyses of P450s, predicted secondary metabolite biosynthetic gene clusters (BGCs), and P450s located in secondary metabolite BGCs. Analysis of the genomes of 114 cyanobacterial species identified 341 P450s in 88 species, belonging to 36 families and 79 subfamilies. In total, 770 secondary metabolite BGCs were found in 103 cyanobacterial species. Only 8% of P450s were found to be part of BGCs. Comparative analyses with other bacteria Bacillus, Streptomyces and mycobacterial species have revealed a lower number of P450s and BGCs and a percentage of P450s forming part of BGCs in cyanobacterial species. A mathematical formula presented in this study revealed that cyanobacterial species have the highest gene-cluster diversity percentage compared to Bacillus and mycobacterial species, indicating that these diverse gene clusters are destined to produce different types of secondary metabolites. The study provides fundamental knowledge of P450s and those associated with secondary metabolism in cyanobacterial species, which may illuminate their value for the pharmaceutical and cosmetics industries.

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Antiplasmodial activity, biosynthetic gene clusters diversity, and secondary metabolite constituent of selected Indonesian Streptomyces

Biodiversitas Journal of Biological Diversity ◽

10.13057/biodiv/d220657 ◽

2021 ◽

Vol 22 (6) ◽

Author(s):

Ema Damayanti ◽

Puspita Lisdiyanti ◽

Andini Sundowo ◽

Shanti Ratnakomala ◽

Achmad Dinoto ◽

...

Keyword(s):

Secondary Metabolites ◽

Ethyl Acetate ◽

Secondary Metabolite ◽

Molecular Identification ◽

Gene Clusters ◽

Antiplasmodial Activity ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Genus Streptomyces ◽

Rdna Sequencing

Abstract. Damayanti E, Lisdiyanti P, Sundowo A, Ratnakomala S, Dinoto A, Widada J, Mustofa. 2021. Antiplasmodial activity, biosynthetic gene clusters diversity, and secondary metabolite constituent of selected Indonesian Streptomyces. Biodiversitas 22: 3478-3487. Actinobacteria of the genus Streptomyces are known as the primary candidate antibiotics, but still limited for antiplasmodial drugs. This study aimed to investigate the antiplasmodial activity, the biosynthetic gene clusters (BGCs) diversity, and the secondary metabolites constituent of selected Indonesian Streptomyces. The bacteria were isolated from various habitats: karst soil (GMR22), mangrove sediments (BSE7F and SHP 22-7), and marine sediment (GMY01). Molecular identification by 16S rDNA sequencing were performed for confirmation and morphological characterization by scanning electron microscope (SEM) were performed for identification. In vitro antiplasmodial assay was performed on human Plasmodium falciparum FCR-3. The BGCs which encode secondary metabolites were analysed using antiSMASH version 5 based on available whole genome sequence (WGS) data. The secondary metabolites were obtained from liquid fermentation followed by extraction using methanol and ethyl acetate. The secondary metabolites constituent was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). The molecular identification showed that GMR22 had similarity to Streptomyces lactacystinicus (98.02%), while BSE7F was similar to Streptomyces althioticus (97.06%), SHP 22-7 was similar to Streptomyces rochei (94.84%), and GMY01 to Streptomyces odonnellii (98.57%). All of isolates had morphological characteristics as the genus Streptomyces bacteria. The highest Plasmodium inhibition (81.84 ± 3.5%) was demonstrated by ethyl acetate extract of marine-derived Streptomyces sp. GMY01 (12.5 µg/mL). Non-ribosomal polyketide synthetase (NRPS), polyketide synthase (PKS) and hybrid of NRPS-PKS were the major BGCs in all Streptomyces. Majority of the Streptomyces produced compounds containing CHON elements with molecular weight approximately 100-400 Da. The active extract of GMY01 bacterium had five major detected compounds, namely kuraramine (C12H18N2O2), laminine (C9H20N2O2) 2-ethylacetanilide (C10H13NO), propoxur (C11H15NO3), and 3-methyl-1,2-diphenylbutan-1-one (C17H18O). This Indonesian marine bacterium is potential for bioassay guided isolation of antiplasmodial compounds in the future studies.

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Understanding the evolutionary origins of bacterial natural products with immunosuppressant properties

10.7287/peerj.preprints.27774v1 ◽

2019 ◽

Author(s):

Wenfa Ng

Keyword(s):

Natural Products ◽

Secondary Metabolites ◽

Secondary Metabolite ◽

Human Body ◽

Immune Cells ◽

Bacterial Species ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Drug Candidates

Actinobacteria and streptomyces are known to produce a variety of natural products, some of which confer antibiotic or immunosuppressive activities. While it is understandable that microbes develop the ability to synthesize molecules such as antibiotics that attack other competing microbes, but why would a secondary metabolite (natural product) synthesized by a microbe confer immunosuppressive activities? Was the capability to synthesize such a molecule endowed by evolution in the context of enabling microbes to develop resistance to immune cells of the human body? Or did the capability come from the need to colonize human body surfaces or gut to gain a survival niche for the microbe? Given that actinobacteria and streptomyces are soil microbes not usually associated with human body surfaces, could their biosynthetic capability for particular immunosuppressants arise from horizontal gene transfer from bacteria that colonize human body surfaces and subsequently develop the ability to synthesize the pertinent compounds through evolution? An alternate line of thinking on this issue touches on the possibility that microbes could encounter analogs of immuno-active molecules in their natural environment. Such molecules might elicit undesired physiological effects on the microbes, which place a selection pressure on microbes to develop countermeasures to the immuno-active molecules through mutations. Hence, through evolution, microbes could have developed the capability to synthesize secondary metabolites able to bind analogs of immuno-active molecules and help sequester them or quench their bioactivity. Subsequent profiling of such secondary metabolites in drug discovery efforts could have uncovered compounds with immunosuppressant activity which are originally developed for counteracting analogs of immuno-active molecules in the environment. It has to be recognized that analogs of immuno-active compounds remain somewhat dissimilar to immune compounds secreted by human immune cells, but they likely share common motifs for protein-secondary metabolite interactions. Direct evidence of the evolution of natural products with immunosuppressant activities could only be obtained from challenging suitable bacterial species with immuno-active molecules. Long cultivation experiments with multiple generations may result in the evolution of biosynthetic gene clusters for the synthesis of natural products able to sequester or quench immuno-active molecules. But, on the another hand, understanding relative binding affinities between a library of natural products and immuno-active molecules from humans would suggest drug candidates and their biosynthetic gene clusters. Subsequent phylogenetic analysis of cluster genes with their homologs from other species may yield insights into the evolution of genes and their putative function.

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Potential secondary metabolite biosynthetic gene clusters and antibacterial activity of novel taxa Gandjariella

Biodiversitas Journal of Biological Diversity ◽

10.13057/biodiv/d211225 ◽

2020 ◽

Vol 21 (12) ◽

Author(s):

Fitria Ningsih ◽

Dhian Chitra Ayu Fitria Sari ◽

Shuhei Yabe ◽

Akira Yokota ◽

Wellyzar Sjamsuridzal

Keyword(s):

Antibacterial Activity ◽

Secondary Metabolites ◽

Secondary Metabolite ◽

Polyketide Synthase ◽

Gene Clusters ◽

Gellan Gum ◽

Biosynthetic Gene ◽

Bacterial Strains ◽

Biosynthetic Gene Clusters ◽

Novel Taxa

Abstract. Ningsih F, Sari DCAF, Yabe S, Yokota A, Sjamsuridzal W. 2020. Potential secondary metabolite biosynthetic gene clusters and antibacterial activity of novel taxa Gandjariella. Biodiversitas 21: 5674-5684. Microbial resistance to available antibiotics has gained increasing attention in recent years and led to the urgent search for active secondary metabolites from novel microbial taxa. This study aimed to assess putative secondary metabolite biosynthetic gene clusters (BGCs) in the genome of a novel thermophilic Actinobacteria type strain Gandjariella thermophila SL3-2-4T and screen for its antibacterial activity. Four other related novel candidate Actinobacteria strains, isolated from forest soil in the Cisolok geothermal area (West Java, Indonesia), were also screened for antibacterial activity in various media solidified with gellan gum. The genome of the SL3-2-4T strain contained 21 antiSMASH-identified secondary metabolite regions harboring BGCs. These BGCs were for polyketide synthase, non-ribosomal peptide synthase, and ribosomally synthesized and post-translationally modified peptide family clusters. Three BGC regions displayed 50-100% similarity with known secondary metabolites. Thirteen and five regions displayed low (4-35%) and no similarity with known BGCs for secondary metabolites, respectively. Strains SL3-2-4T and SL3-2-7 on MM 2 medium solidified with gellan gum at 45 °C for 14 days demonstrated inhibitory activity against all Gram-positive, but not Gram-negative bacteria. Strain SL3-2-10 on ISP 3 gellan gum medium incubated for seven days only active against K. rhizophila NBRC 12078. Strains SL3-2-6 and SL3-2-9 did not exhibit any antibacterial activity against the tested bacterial strains on the three tested media. The results indicated that novel taxa have the potential for the discovery of active secondary metabolites.

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Boosting Secondary Metabolite Production and Discovery through the Engineering of Novel Microbial Biosensors

BioMed Research International ◽

10.1155/2018/7021826 ◽

2018 ◽

Vol 2018 ◽

pp. 1-11 ◽

Cited By ~ 6

Author(s):

Ulysses Amancio de Frias ◽

Greicy Kelly Bonifacio Pereira ◽

María-Eugenia Guazzaroni ◽

Rafael Silva-Rocha

Keyword(s):

Secondary Metabolites ◽

Secondary Metabolite ◽

Gene Clusters ◽

Secondary Metabolite Production ◽

Special Focus ◽

Biosynthetic Gene ◽

Expression Systems ◽

Biosynthetic Gene Clusters ◽

Metabolite Production ◽

Synthetic Promoters

Bacteria are a source of a large number of secondary metabolites with several biomedical and biotechnological applications. In recent years, there has been tremendous progress in the development of novel synthetic biology approaches both to increase the production rate of secondary metabolites of interest in native producers and to mine and reconstruct novel biosynthetic gene clusters in heterologous hosts. Here, we present the recent advances toward the engineering of novel microbial biosensors to detect the synthesis of secondary metabolites in bacteria and in the development of synthetic promoters and expression systems aiming at the construction of microbial cell factories for the production of these compounds. We place special focus on the potential of Gram-negative bacteria as a source of biosynthetic gene clusters and hosts for pathway assembly, on the construction and characterization of novel promoters for native hosts, and on the use of computer-aided design of novel pathways and expression systems for secondary metabolite production. Finally, we discuss some of the potentials and limitations of the approaches that are currently being developed and we highlight new directions that could be addressed in the field.

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Comparative Genomics Determines Strain-Dependent Secondary Metabolite Production in Streptomyces venezuelae Strains

Biomolecules ◽

10.3390/biom10060864 ◽

2020 ◽

Vol 10 (6) ◽

pp. 864

Author(s):

Woori Kim ◽

Namil Lee ◽

Soonkyu Hwang ◽

Yongjae Lee ◽

Jihun Kim ◽

...

Keyword(s):

Secondary Metabolites ◽

Secondary Metabolite ◽

Gene Clusters ◽

Secondary Metabolite Production ◽

Biosynthetic Gene ◽

Genome Sequences ◽

Biosynthetic Gene Clusters ◽

Streptomyces Venezuelae ◽

Metabolite Production ◽

Non Coding Rna

Streptomyces venezuelae is well known to produce various secondary metabolites, including chloramphenicol, jadomycin, and pikromycin. Although many strains have been classified as S. venezuelae species, only a limited number of strains have been explored extensively for their genomic contents. Moreover, genomic differences and diversity in secondary metabolite production between the strains have never been compared. Here, we report complete genome sequences of three S. venezuelae strains (ATCC 10712, ATCC 10595, and ATCC 21113) harboring chloramphenicol and jadomycin biosynthetic gene clusters (BGC). With these high-quality genome sequences, we revealed that the three strains share more than 85% of total genes and most of the secondary metabolite biosynthetic gene clusters (smBGC). Despite such conservation, the strains produced different amounts of chloramphenicol and jadomycin, indicating differential regulation of secondary metabolite production at the strain level. Interestingly, antagonistic production of chloramphenicol and jadomycin was observed in these strains. Through comparison of the chloramphenicol and jadomycin BGCs among the three strains, we found sequence variations in many genes, the non-coding RNA coding regions, and binding sites of regulators, which affect the production of the secondary metabolites. We anticipate that these genome sequences of closely related strains would serve as useful resources for understanding the complex secondary metabolism and for designing an optimal production process using Streptomyces strains.

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