Lincomycin at Subinhibitory Concentrations Potentiates Secondary Metabolite Production by Streptomyces spp

Abstract

e Antibiotics have either bactericidal or bacteriostatic activity. However, they also induce considerable gene expression in bacteria when used at subinhibitory concentrations (below the MIC). We found that lincomycin, which inhibits protein synthesis by binding to the ribosomes of Gram-positive bacteria, was effective for inducing the expression of genes involved in secondary metabolism in Streptomyces strains when added to medium at subinhibitory concentrations. In Streptomyces coelicolor A3(2), lincomycin at 1/10 of its MIC markedly increased the expression of the pathway-specific regulatory gene actII-ORF4 in the bluepigmented antibiotic actinorhodin (ACT) biosynthetic gene cluster, which resulted in ACT overproduction. Intriguingly, S. lividans 1326 grown in the presence of lincomycin at a subinhibitory concentration (1/12 or 1/3 of its MIC) produced abundant antibacterial compounds that were not detected in cells grown in lincomycin-free medium. Bioassay and mass spectrometry analysis revealed that some antibacterial compounds were novel congeners of calcium-dependent antibiotics. Our results indicate that lincomycin at subinhibitory concentrations potentiates the production of secondary metabolites in Streptomyces strains and suggest that activating these strains by utilizing the dose-response effects of lincomycin could be used to effectively induce the production of cryptic secondary metabolites. In addition to these findings, we also report that lincomycin used at concentrations for markedly increased ACT production resulted in alteration of the cytoplasmic protein (F o F 1 ATP synthase ␣ and ␤ subunits, etc.) profile and increased intracellular ATP levels. A fundamental mechanism for these unique phenomena is also discussed. Streptomyces is the largest genus of Gram-positive filamentous actinomycetes, and members of this genus produce abundant amounts of numerous bioactive metabolites, including antitumor agents, immunosuppressants, and antibiotics in particular (1, 2). Whole-genome sequencing studies of Streptomyces strains have shown that each species could produce many more secondary metabolites than were expected. For example, Streptomyces coelicolor A3(2), S. avermitilis MA-4680, and S. griseus IFO 13350 each produce several secondary metabolites, although they have more than 20 gene clusters that can encode a number of known or predicted biosynthetic pathways for secondary metabolites (3-5). This indicates that the vast majority of secondary metabolites remain unexpressed or barely expressed under standard laboratory conditions. Thus, there is considerable interest in exploring practical means to induce this genetic potential in streptomycetes, which could result in the isolation of novel bioactive secondary metabolites.Recent developments in new methodologies, including physiological and genetic engineering approaches, have opened the door for the discovery of novel secondary metabolites by activating cryptic biosynthetic pathways in streptomycetes (6-13). The improvements and modifications of ...

Antibiotic dialogues: induction of silent biosynthetic gene clusters by exogenous small molecules

“…This was the case with tetracycline and lincomycin (Shima et al 1996;Imai et al 2015). It will be interesting to see what the mechanism of induced secondary metabolite production is in these non-resistant strains, a case that is akin to examples of goadsporin or promomycin in coculture experiments.…”

Elicitation of secondary metabolism in actinomycetes

“…Antibiotics at sublethal concentrations can generate diverse metabolic responses in bacteria (Imai et al, 2015). Identification of elicitors that induce the expression of silent gene clusters under standard conditions is however a challenge.…”

Identification of a Novel Lincomycin Resistance Mutation Associated with Activation of Antibiotic Production in Streptomyces coelicolor A3(2)

“…After induction at 37°C for 4 h, the cells were collected by centrifugation and resuspended in fresh LB broth to an OD 600 of ϳ1.0. A 20-l aliquot of each was inoculated onto LB plates containing ampicillin (100 g/ml), IPTG (50 g/ml), and lincomycin (200 g/ml), followed by incubation at 37°C for 24 h. centrations of lincomycin (1/16 to 1/4 of its MIC) in the culture medium markedly enhanced ACT production by S. coelicolor (38), although the mechanism underlying this ACT overproduction appears different from that of the two-component system. Thus, the acquisition of lincomycin resistance by certain spontaneous mutations and the addition of lincomycin into the medium both could be effective at activating or enhancing antibiotic production in actinomycetes.…”