19 20 21 22 23 101 10% (v/v) glycerol and serial dilutions were spread onto either soya flour mannitol (SFM) agar, 102 starch casein agar, or minimal medium agar containing sodium citrate (Lebeis et al 2015).103Plates were incubated at 30C for up to 14 days. Colonies resembling streptomycetes were re-104 streaked onto SFM agar and identified by 16S rRNA gene PCR amplification and sequencing 105 with universal primers PRK341F and MPRK806R (see Table S1 for primers, plasmids and 106 strains used in this work). Streptomyces strains were maintained on SFM agar (N1, N2, M2, 107 M3 and S. coelicolor M145), Maltose/Yeast extract/Malt extract (MYM) agar with trace 108 5 elements (L2) or ISP2 agar (S. lydicus strains); media recipes are detailed in Table S2. Spore 109 stocks were made as described previously (Kieser et al 2000). 110 111 Genome sequencing and analysis. High quality genome sequences were obtained for newly 112 isolated strains N1, N2, M2, M3, and L2, as well as three known strains of Streptomyces 113 lydicus; one isolated from the commercial plant growth-promoting product Actinovate and two 114 additional S. lydicus strains (ATCC25470 and ATCC31975) obtained from the American Type 115 Culture Collection. Strains were sequenced using PacBio RSII sequencing technology at the 116 131 isolated from a patient at the Norfolk and Norwich University Hospital UK (Qin et al 2017), 132Escherichia coli and Pseudomonas syringae DC3000 were grown overnight in 10 ml Lysogeny 133 Broth (LB) (1% tryptone, 0.5% NaCl, supplemented with 0.1% glucose for P. syringae). These 134 were sub-cultured 1 in 20 (v/v) for a further 4 hours at 30°C for P. syringae and 37°C for E. 135 coli. These cultures were then used to inoculate 100 ml of molten LB (0.5% agar, plus 0.1% 136 glucose for P. syringae), of which 3 ml was used to overlay agar plates containing Streptomyces 137 colonies. Plates were incubated for 48 hours at 30C, and bioactivity was indicated by a clear 138 halo around the Streptomyces colony. For bioassays using the fungal strains Lomentospora 139 prolificans or Gaeumannomyces graminis, Streptomyces species were grown for 7 days, and 140 then a plug of the fungus (grown on potato glucose agar, Sigma Aldrich, for 14 days) was 141
Streptomyces bacteria make numerous secondary metabolites, including half of all 25 known antibiotics. Production is coordinated with their complex life cycles but the regulators 26 that coordinate development with antibiotic biosynthesis are largely unknown. This is 27 important to understand because most Streptomyces secondary metabolites are not produced 28 under laboratory conditions and unlocking the 'cryptic' biosynthetic gene clusters (BGCs) is a 29 major focus for antibiotic discovery. Here we characterise the highly conserved actinobacterial 30 response regulator MtrA in Streptomyces species. MtrA is essential and regulates cell cycle 31 progression in Mycobacterium tuberculosis. We show that MtrA is also essential in 32 Streptomyces venezuelae where it controls genes required for DNA replication and cell 33 division. MtrA also directly regulates the expression of genes in >70% of its BGCs and 34 artificially activating MtrA switches on the production of antibiotics in S. coelicolor and S.
Cuticular microbiomes of Acromyrmex leaf-cutting ants are exceptional because they are freely colonizable, and yet the prevalence of Pseudonocardia, a native vertically transmitted symbiont that controls Escovopsis fungus-garden disease, is never compromised. Game theory suggests that competition-based screening can allow the selective recruitment of antibiotic-producing bacteria from the environment, by fomenting and biasing competition for abundant host resources. Mutual symbiont aggression benefits the host and also maintains native symbiont viability. Here we use RNA-stable isotope probing (RNA-SIP) to confirm predictions that Acromyrmex cuticles can maintain a range of microbial symbionts. We then used dual-RNA-sequencing and bioassays to show that vertically transmitted Pseudonocardia strains produce antibacterials that differentially reduce the growth rates of other microbes, ultimately eliminating non-antibiotic-producing strains that might parasitize the symbiosis while still allowing antibiotic-producing Streptomyces strains to survive. Open cuticular microbiomes can thus maintain a specific co-evolved mutualism by restricting access for other bacterial strains.
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