Response of Secondary Metabolism of Hypogean Actinobacterial Genera to Chemical and Biological Stimuli

Abstract

Microorganisms within microbial communities respond to environmental challenges by producing biologically active secondary metabolites, yet the majority of these small molecules remain unidentified. We have previously demonstrated that secondary metabolite biosynthesis in actinomycetes can be activated by model environmental chemical and biological stimuli, and metabolites can be identified by comparative metabolomics analyses between stimuli conditions. Herein, we survey the secondary metabolite productivity of a group of 20 phylogenetically diverse actinobacteria isolated from hypogean (cave) environments by applying a battery of stimuli consisting of exposure to antibiotics, metals, and mixed microbial culture. Comparative metabolomics was used to reveal secondary metabolite responses from stimuli. These analyses revealed substantial changes in global metabolomic dynamics with over 30 % of metabolomic features increasing more than 10-fold under at least one stimulus condition. Selected features were isolated and identified via NMR, revealing several known secondary metabolite families including the tetarimycins, aloesaponarins, hypogeamicins, actinomycins, and propeptins. One prioritized metabolite was identified to be a previously unreported aminopolyol polyketide, funisamine, produced by a cave isolate when exposed to mixed culture. The production of funisamine was most significantly increased in mixed culture with sp. The biosynthetic gene cluster responsible for the production of funisamine was identified via genomic sequencing of the producing strain, sp. KDCAGE35, which facilitated deduction of its biosynthesis. Together, these data demonstrate that comparative metabolomics can reveal the stimulus-induced production of natural products from diverse microbial phylogenies. Microbial secondary metabolites are an important source of biologically active and therapeutically relevant small molecules. However, much of this active molecular diversity is challenging to access due to low production levels or difficulty in discerning secondary metabolites within complex microbial extracts prior to isolation. Herein we demonstrate that ecological stimuli increase secondary metabolite production in phylogenetically diverse actinobacteria isolated from understudied hypogean environments. Additionally, we show that comparative metabolomics linking stimuli to metabolite response data can effectively reveal secondary metabolites within complex biological extracts. This approach highlighted secondary metabolites in almost all observed natural product classes, including low abundance analogs of biologically relevant metabolites, as well as a new linear aminopolyol polyketide, funisamine. This study demonstrates the generality of activating stimuli to potentiate secondary metabolite production across diverse actinobacterial genera.

Cave Actinobacteria as Producers of Bioactive Metabolites

“…Moreover, isolation media that mimic the conditions of low concentration nutrients in caves such as tap water agar, 1/100 ISP2 and oligotrophic medium (M5) were also successfully used for the isolation of actinobacteria. (Lee et al, 2000b; Velikonja et al, 2014; Covington et al, 2018; Passari et al, 2018). High concentration of nutrients in standard cultivation media were reported to cause cell death in cave-associated bacteria due to osmotic stress (Barton, 2006; Ghosh et al, 2017).…”

“…Therefore, the second challenge lies in our ability to activate these silent gene clusters. Recently, specific biological and chemical stimuli namely exposure to antibiotics, metals and mixed microbial culture, were successfully employed to activate secondary metabolites production in cave actinobacteria (Covington et al, 2018). Evidently, the study on cave actinobacteria and their bioactive compounds is still at an early stage.…”

Antibacterial potential of Actinobacteria from a Limestone Mining Site in Meghalaya, India

“…Limestone-rich areas mainly dominated by a CaCO 3 environment, are under-explored ecological niches for mining of microorganisms for various metabolic properties. Recent studies have revealed that both hypogean and epigean limestone actinobacteria are potential antibiotics producers [8][9][10][11][12][13][14] . The state of Meghalaya located in the north-eastern part of India falls under the Indo-Burma Biodiversity Hotspot 15 .…”

Genome-based classification of the Streptomyces violaceusniger clade and description of Streptomyces sabulosicollis sp. nov. from an Indonesian sand dune

“…In contrast, only the genomes of the isolate and the type strains of S. iranensis , S. violaceusniger and S. rapamycinicus contained bioclusters considered to express for meilingmycin, an anti-parasitic macrolide (Sun et al 2002 ) and nigericin, which inhibits Gram-positive bacteria (Graven et al 1966 ). Similarly, the draft genomes of the isolate, S. albiflaviniger DSM 41598 T and S. javensis DSM 41764 T contained bioclusters predicted to encode for the synthesis of cahuitamycins A-C, which inhibit the formation of bacterial biofilms (Park et al 2016 ), pladienolides, anti-tumour antibiotics (Mizui et al 2004 ) and funisamine, an aminopolyol polyketide antibiotic which inhibits the growth of wild type strains of Staphylococcus aureus , Escherichia coli and Candida albicans (Covington et al 2018 ), respectively. Bioclusters predicted to encode for rapamycin were only detected in the genomes of the S. iranensis and S. rapamycinicus strains.…”