Lincomycin at Subinhibitory Concentrations Potentiates Secondary Metabolite Production by Streptomyces spp

Abstract

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 blue-pigmented 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 (FoF1 ATP synthase α and β subunits, etc.) profile and increased intracellular ATP levels. A fundamental mechanism for these unique phenomena is also discussed.

FIG 1

S. coelicolor A3(2) and S. lividans ACT production and colony morphologies. Strains 1147 and 1326 were inoculated onto MR5 agar that contained clindamycin (Cli), chloramphenicol (Cm), erythromycin (Ery), gentamicin (Gen), lincomycin (Lin), streptomycin (Sm), tetracycline (Tet), thiostrepton (Tsp), or tylosin (Tyl) at the concentrations indicated and then incubated at 30°C for 7 and 10 days, respectively. (A) Results in the presence of ribosome-targeting antibiotics. (B) Results in the presence of different lincomycin concentrations.

FIG 2

Effects of lincomycin on S. coelicolor A3(2) antibiotic production and biomass in liquid culture. Strain 1147 was inoculated into MR5 liquid medium that contained different lincomycin concentrations and then incubated at 30°C for 10 days with shaking (200 rpm). Pigmented antibiotic (ACT and RED) production and dry cell weight were determined as described in Materials and Methods. All experiments were done in triplicate, and results are expressed as means ± standard deviations.

FIG 3

S. coelicolor A3(2) growth, ACT production, and expression of the pathway-specific regulatory genes in the ACT and RED biosynthetic gene clusters with and without lincomycin. (A) Growth and ACT production in liquid medium. Strain 1147 was inoculated into MR5 liquid medium with or without lincomycin (10 μg/ml) and then incubated at 30°C on a rotary shaker. Growth was monitored by measuring dry cell weight. Biomass results at the end of the culture period (240 h) were as follows: without lincomycin, 2.23 mg of dry cell wt/ml; with lincomycin, 2.37 mg of dry cell wt/ml. At the times indicated by arrows, E (mid-exponential phase), T (transition phase), S1 (16 h after T, early stationary phase), S2 (96 h after T, late stationary phase), and S3 (144 h after T, late stationary phase), cells were harvested to prepare total RNA and total soluble cellular proteins. ACT production was determined as described above. (B) Transcriptional analysis of actII-ORF4 and redD by real-time qRT-PCR. RNA was extracted from cells that had grown to the growth phases indicated. Total RNA preparation and real-time qRT-PCR were performed as described in Materials and Methods. Maximum expression levels were compared after setting the maximum expression level of a control (the exponential phase without lincomycin) to 1. (C) Western blot analysis for ActII-ORF4 expression. Protein samples were prepared from cells that had grown to the growth phases indicated. Western blot analysis was performed as described in Materials and Methods. Each lane was loaded with 5 μg of total soluble cellular proteins.

FIG 4

Antibacterial compounds and transcriptional analysis of the gene for CDA biosynthesis in S. lividans grown with lincomycin at subinhibitory concentrations. Strain 1326 was inoculated onto MR5 agar plates that contained different concentrations of lincomycin and then incubated at 30°C for 7 days. (A) Antibacterial compound production. Agar plugs were prepared from agar plates, transferred to an assay plate inoculated with a lincomycin-resistant mutant of B. subtilis ATCC 6633 as a test organism, and then incubated at 37°C for 24 h. (B) HPLC analysis of culture extracts. Samples were prepared and analyzed by HPLC as described in Materials and Methods. Eluate absorbance was monitored at 220 nm. Arrows indicate peaks that were barely detectable in samples prepared from an agar plate without lincomycin. The insert shows the antibacterial activities of peaks 1 and 2 against B. subtilis ATCC 6633 in the presence or absence of 12 mM calcium nitrate. (C) Transcriptional analysis of SLI3570 by real-time qRT-PCR. Cells were harvested to prepare total RNA at phase 1 (during substrate mycelia formation), phase 2 (just when RED production began), phase 3 (12 h after phase 2), and phase 4 (24 h after phase 2). Real-time qPCR was performed as described in Materials and Methods.

FIG 5

Total cellular protein profiles, transcriptional analysis of the gene coding for F_oF₁ ATP synthase, and changes in intracellular nucleotide levels in S. coelicolor A3(2) grown with or without lincomycin. (A) SDS-PAGE analysis of total cellular proteins. Total cellular protein samples were prepared from cells grown to the indicated growth phases in MR5 medium without (−) or with (+) lincomycin (10 μg/ml). Each lane was loaded with 5 μg of protein. Molecular mass markers (Sigma low-range molecular weight markers) are shown at the left. Arrows 2, 3, and 6 indicate proteins that were abundant in a sample prepared from cells grown with lincomycin but were minimal in samples prepared from cells grown without lincomycin, and arrows 1, 4, and 5 indicate the opposite conditions. Protein bands 1 to 6 were identified by peptide mass fingerprinting analysis. (B) Transcriptional analysis of atpA and atpD by real-time qRT-PCR. Cells were harvested to prepare total RNA as described in the legend to Fig. 3A. Real-time qRT-PCR was performed as described in Materials and Methods. (C) Intracellular nucleotide level changes. Strain 1147 was inoculated in MR5 liquid medium with or without lincomycin (10 μg/ml) and then incubated at 30°C on a rotary shaker. At the growth phases indicated by arrows E, T, S1, and S2 in Fig. 3A, samples were prepared and analyzed by HPLC as described in Materials and Methods. All experiments were done in triplicate, and results are expressed as means ± standard deviations.

FIG 6

Streptomycin production by S. griseus in liquid culture. Strain IFO 13189 was inoculated into SPY liquid medium that contained different concentrations of lincomycin and then incubated at 25°C with shaking for 4 days. Streptomycin production was determined as described in Materials and Methods.

FIG 7

Cross-culture of S. coelicolor A3(2) with S. erythraea or S. griseus. S. coelicolor A3(2) 1147 (horizontal line) was cross-cultured with S. erythraea NRRL 2338 (A) or S. griseus IFO 13189 (B) (vertical line) on MR5 agar at 30°C for 8 days. Blue color represents the antibiotic ACT.