Multifactorial genetic control and magnesium levels govern the production of a Streptomyces antibiotic with unusual cell density dependence

Abstract

Streptomyces bacteria are renowned both for their antibiotic production capabilities and for their cryptic metabolic potential. Their metabolic repertoire is subject to stringent genetic control, with many of the associated biosynthetic gene clusters being repressed by the conserved nucleoid-associated protein Lsr2. In an effort to stimulate new antibiotic production in wild Streptomyces isolates, we leveraged the activity of an Lsr2 knockdown construct and successfully enhanced antibiotic production in the wild Streptomyces isolate WAC07094. We determined that this new activity stemmed from increased levels of the angucycline-like family member saquayamycin. Saquayamycin has both antibiotic and anti-cancer activities, and intriguingly, beyond Lsr2-mediated repression, we found saquayamycin production was also suppressed at high density on solid or in liquid growth media; its levels were greatest in low-density cultures. This density-dependent control was exerted at the level of the cluster-situated regulatory gene sqnR and was mediated in part through the activity of the PhoRP two-component regulatory system, where deleting phoRP led to both constitutive antibiotic production and sqnR expression. This suggests that PhoP functions to repress the expression of sqnR at high cell density. We further discovered that magnesium supplementation could alleviate this density dependence, although its action was independent of PhoP. Finally, we revealed that the nitrogen-responsive regulators GlnR and AfsQ1 could relieve the repression exerted by Lsr2 and PhoP. Intriguingly, we found that this low density-dependent production of saquayamycin was not unique to WAC07094; saquayamycin production by another wild isolate also exhibited low-density activation, suggesting that this spatial control may serve an important ecological function in their native environments.IMPORTANCEStreptomyces specialized metabolic gene clusters are subject to complex regulation, and their products are frequently not observed under standard laboratory growth conditions. For the wild Streptomyces isolate WAC07094, production of the angucycline-family compound saquayamycin is subject to a unique constellation of control factors. Notably, it is produced primarily at low cell density, in contrast to the high cell density production typical of most antibiotics. This unusual density dependence is conserved in other saquayamycin producers and is driven by the pathway-specific regulator SqnR, whose expression is influenced by both nutritional and genetic elements. Collectively, this work provides new insights into an intricate regulatory system governing antibiotic production and indicates there may be benefits to including low-density cultures in antibiotic screening platforms.

Keywords: Streptomyces; antibiotic; cell density; magnesium; phosphate; regulation.

Fig 1

Saquayamycin production by Streptomyces sp. WAC07094. (A) Antibiotic bioassay in which wild type (WT) WAC07094 and Lsr2 knockdown (K/D) strains (overexpressing a dominant negative variant of lsr2) were spotted to Bennett’s medium, grown for 3 days, and then overlaid with the sensitive indicator strains Bacillus subtilis (left), methicillin-resistant Staphylococcus aureus (middle), or vancomycin-resistant Enterococcus faecium (right). (B) Schematic diagram of the saquayamycin biosynthetic cluster in Streptomyces KY40-1 (bottom), compared with the equivalent cluster from WAC07094 (top). Genes from the WAC07094 strain studied here are indicated with hashed lines. (C) Antibiotic bioassay using B. subtilis as the indicator strain, together with crude extracts spotted to filter disks, from the sqnHI disruption mutant and the sqnR overexpression strains (both carrying the Lsr2 knockdown construct) grown for 3 days on Bennett’s medium, compared with their relevant controls. (D) Liquid chromatography-mass spectrometry analysis of crude extracts prepared from WAC07094 grown for 3 days on Bennett’s agar (agar + biomass). (Left) Chromatograms of [M-H]⁻ ions extracted at m/z of 819; (right) mass spectra of the peak at m/z of 819 and its assigned chemical formula; (bottom) structures of saquayamycin A and B.

Fig 2

Saquayamycin activity and sqnR expression are density dependent. (A) (Left) Agar plugs were excised from the center and edge of a confluent lawn of the WAC07094 Lsr2 knockdown strain; (right) bioassays for antibiotic production, conducted by removing agar plugs (from the colony center or edge, as indicated on the left) of the Lsr2 knockdown strain, placing them onto B. subtilis-containing medium and incubating the plates overnight at 37°C. (B) Chromatograms (UV) of crude extracts prepared from Bennett’s agar on which the Lsr2 knockdown strain (left) and same strain carrying the sqnR overexpressing construct (right) had been grown as a lawn for 3 days. Peaks of saquayamycin A and B are indicated with the A and B labels, respectively. (Inset) Anti-Bacillus activity exerted by corresponding wild type (left) and sqnR-overexpressing (right) strains, where their associated agar plugs were taken from the center of a confluent lawn (high density). (C) (Top) Antibiotic bioassay of the Lsr2 knockdown strain carrying either the promoter-less gfp (left) or sqnR promoter-driven gfp construct, against B. subtilis. Zones of clearing indicate antibiotic activity; (bottom) promoter activity is seen as green fluorescence produced by the same Streptomyces strains, where red signals represent saturated fluorescence intensities (high promoter activity).

Fig 3

Density-dependent repression of saquayamycin production is alleviated by magnesium supplementation. (A) Antibiotic bioassay of wild type (WT) and Lsr2 knockdown (K/D) strains (carrying the sqnR transcriptional reporter) grown for 3 days on Bennett’s medium in the absence (left) or presence (right) of magnesium supplementation, where the resulting lawns of the two strains were overlaid with soft agar infused with B. subtilis. Zones of clearing indicate antibiotic activity. (B) sqnR promoter activity of the strains shown in panel A, as measured by green fluorescence of different biological replicates.

Fig 4

The density-dependent expression of mtpA and the saquayamycin cluster is affected by magnesium and PhoRP. (A) Promoter activity of mtpA in wild-type WAC07094 after 3 days’ growth on Bennett’s agar with or without magnesium chloride, manganese chloride, or zinc chloride supplementation. (B) Genetic organization of the mtpA, phoU, phoR, and phoP locus. Transcription start sites are indicated by vertical lines, with the line widths (and associated horizontal arrows) approximating relative transcript abundance. Beneath the genes are four lines, indicating the sequence included in each of four complementation constructs (labeled I–IV on the right). (C) Antibiotic activity as detected using B. subtilis infused agar, overlaying lawns of wild type (WT) or mutant strains grown for 3 days on Bennett’s agar, with or without additional magnesium supplementation. (D) Promoter activity of sqnR after 3 days of growth, as indicated by the relative fluorescence within the lawn of the wild type and phoRP locus mutant.

Fig 5

GlnR positively controls saquayamycin production. (A) Schematic of the sqnR promoter region, including the transcription start site (+1), the upstream promoter (−10 and −35), and the predicted binding sites for Lsr2, PhoP, MtrA, GlnR, and AfsQ1. The promoter region used for the reporter constructs is indicated in purple. (B) Antibiotic bioassay using WAC07094 wild type (WT) or Lsr2 knockdown (K/D) strains carrying either the empty plasmid pIJ10257 (control), or glnR or mtrA under the highly active ermE* promoter, or afsQ1* under the inducible tipA promoter. B. subtilis was used as the sensitive indicator strain, and growth inhibition zones were quantified and normalized to the corresponding Streptomyces growth area. Error bars indicate standard deviation (n = 4). (C) The effect of phoRP deletion relative to Lsr2 knockdown, and Lsr2 knockdown together with glnR overexpression, were assessed using B. subtilis-infused agar overlays. The darker coloration associated with the two rightmost strains indicates full growth inhibition, in contrast to inhibition only at the periphery seen for the leftmost plate.

Fig 6

The density dependence of saquayamycin production is conserved across diverse streptomycetes. (A) Antibiotic bioassays were conducted using B. subtilis as the sensitive indicator organism, together with extracts of wild type (WT) and Lsr2 knockdown (K/D) WAC07094 cultures inoculated from low-density (10³/mL), mid-density (10⁵/mL) and high-density (10⁷ /mL) spore preparations and grown for 4 days in Bennett’s liquid medium. (B) Experiments equivalent to that in panel A were conducted for Streptomyces isolate CCESR44 wild type and Lsr2 knockdown strains, including only an additional low-density inoculum of 100–200 spores/mL. (C) Extracted ion chromatograms of [M-H]⁻ (at m/z 819.3) from metabolite profiles for low- and high-cell density inoculated cultures from panel B.

Fig 7

Proposed model of the regulatory and nutritional inputs impacting saquayamycin production, both highlighting known features of the system and acknowledging the possibility of additional (as yet unknown) contributing systems.