Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2)

Abstract

A strain of Streptomyces lividans, TK24, was found to produce a pigmented antibiotic, actinorhodin, although S. lividans normally does not produce this antibiotic. Genetic analyses revealed that a streptomycin-resistant mutation str-6 in strain TK24 is responsible for induction of antibiotic synthesis. DNA sequencing showed that str-6 is a point mutation in the rpsL gene encoding ribosomal protein S12, changing Lys-88 to Glu. Gene replacement experiments with the Lys883Glu str allele demonstrated unambiguously that the str mutation is alone responsible for the activation of actinorhodin production observed. In contrast, the strA1 mutation, a genetic marker frequently used for crosses, did not restore actinorhodin production and was found to result in an amino acid alteration of Lys-43 to Asn. Induction of actinorhodin production was also detected in strain TK21, which does not harbor the str-6 mutation, when cells were incubated with sufficient streptomycin or tetracycline to reduce the cell's growth rate, and 40 and 3% of streptomycin-or tetracycline-resistant mutants, respectively, derived from strain TK21 produced actinorhodin. Streptomycin-resistant mutations also blocked the inhibitory effects of relA and brgA mutations on antibiotic production, aerial mycelium formation or both. These str mutations changed Lys-88 to Glu or Arg and Arg-86 to His in ribosomal protein S12. The decrease in streptomycin production in relC mutants in Streptomyces griseus could also be abolished completely by introducing streptomycin-resistant mutations, although the impairment in antibiotic production due to bldA (in Streptomyces coelicolor) or afs mutations (in S. griseus) was not eliminated. These results indicate that the onset and extent of secondary metabolism in Streptomyces spp. is significantly controlled by the translational machinery.

Discovery of Unusual Biaryl Polyketides by Activation of a SilentStreptomyces venezuelaeBiosynthetic Gene Cluster

“…MYM‐TAP liquid medium (with 400 μL of trace element solution44 added per 200 mL of medium added after autoclaving) was used to grow Streptomyces strains for venemycin production and for RNA isolation from S. coelicolor M1825. For the halogenation studies we used Minimal Medium (MM)44 supplemented with NaCl or NaBr (0.5 g L −1 ), and modified versions of R3 medium45 consisting of glucose (10 g L −1 ), yeast extract (5 g L −1 ), casamino acids (100 mg L −1 ), proline (3 g L −1 ), MgSO 4 ⋅ 7 H 2 O (10 g L −1 ), CaSO 4 ⋅ 2 H 2 O (4 g L −1 ), K 2 SO 4 (200 mg L −1 ), KH 2 PO 4 (50 mg L −1 ), TES [ N ‐tris(hydroxymethyl)methyl‐2‐aminoethanesulfonic acid, 5.6 g L −1 ], trace elements (as described for R2YE medium44) and NaCl or NaBr or both (1 g L −1 ); the media were adjusted to pH 7.2 with NaOH and autoclaved. Streptomyces liquid cultures were performed in medium (50 mL) in 250 mL flasks containing stainless steel springs to promote dispersed growth with at least two replicates.…”

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

“…Here we focus on the original studies that provided the impetus for the field as well as a molecular rationale for ribosome engineering. As alluded to above, wt S. lividans TK21 normally does not produce Act, even though it contains a complete act biosynthetic gene cluster (Shima et al 1996). The original study by Ochi and colleagues investigated how the streptomycinresistant S. lividans strain TK24 produced abundant quantities of Act (Fig.…”

“…The inventors of the method were inspired by the observation that a mutated, streptomycin-resistant strain of Streptomyces lividans produced the blue pigment actinorhodin (Act, Fig. 1, 2), whereas the parent strain, from which the mutant was derived, did not (Shima et al 1996). They mapped the mutation to a ribosomal protein and showed that only the mutant ribosome led to activation of the silent Act gene cluster (act), thus demonstrating translational control in Act biosynthesis.…”

Guanine Nucleotides Guanosine 5′-Diphosphate 3′-Diphosphate and GTP Co-operatively Regulate the Production of an Antibiotic Bacilysin in Bacillus subtilis

“…Working with the genera Streptomyces and Bacillus, the correlation between the stringent response and antibiotic production has been established. In the absence of a functional relA or rplK gene, antibiotic production by these organisms is remarkably reduced or lost (11)(12)(13)(14). Therefore, the initiation of antibiotic production is considered to be positively regulated by the stringent response (15-18), * This work was supported by a grant from the Organized Research Combination System of the Science and Technology Agency of Japan.…”