Genetic and physiological characterization of rpoB mutations that activate antibiotic production in Streptomyces lividans

Abstract

Antibiotic production in Streptomyces lividans can be activated by introducing certain mutations (rif) into the rpoB gene that confer resistance to rifampicin. Working with the most typical (rif-17) mutant strain, KO-417, the rif-17 mutation was characterized. The rif-17 mutation was shown to be responsible for activating antibiotic production and for reducing the growth rate of strain KO-417, as demonstrated by gene-replacement experiments. Gene-expression analysis revealed that introduction of rif into S. lividans elevates expression of the pathway-specific regulatory gene actII-ORF4 to nearly the same level seen in Streptomyces coelicolor. The rif effect on antibiotic production was still evident in the genetic background of relC, indicating that the rif mutation can provoke its effect without depending on ppGpp. Accompanying the restoration of antibiotic production, rif mutants also exhibited a lower rate of RNA synthesis compared to the parental strain when grown in a nutritionally rich medium, suggesting that the mutant RNA polymerases may behave like 'stringent' RNA polymerases. These results indicate that the rif mutation can alter the gene-expression pattern independent of ppGpp. The impaired growth of strain KO-417 (rif-17) was largely restored by introducing the second rif mutation (rif-18) just adjacent to the rif-17 position. Proteome analysis using two-dimensional PAGE revealed that the rif mutant strain KO-418 (rif-17 rif-18) displayed a temporal burst of expression especially of two enzymes, glutamine synthetase (type II) and oxidoreductase, during the late growth phase.

RNA Polymerase Mutation Activates the Production of a Dormant Antibiotic 3,3′-Neotrehalosadiamine via an Autoinduction Mechanism in Bacillus subtilis

“…Similarly, in S. coelicolor A3(2) and S. lividans, alteration at His-437 (corresponding to His-482 in B. subtilis) to Arg or Tyr can suppress the deficiency of antibiotic production resulting from the lack of ppGpp due to relA or relC mutation (9 -11). These results suggest that the mutant RNAPs functionally mimic "stringent" RNAP, leading to the activation of a stringent response-dependent antibiotic biosynthesis pathway (10,11). Although rpoB5 mutation also effectively activated NTD production, the activation mechanism for NTD production in B. subtilis appears quite different from that in Streptomyces spp.…”

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

“…Among these, experiments with rifampicin have been the most successful (Hu, Zhang and Ochi 2002;Lai et al 2002;Xu et al 2002). Rifampicin (Fig.…”

Activation of Dormant Secondary Metabolism Neotrehalosadiamine Synthesis by an RNA Polymerase Mutation inBacillus subtilis

“…Jørgensen and his co-workers also found that several rifampicin-resistance mutations (including the S487L mutation) resulted in increased production of extracellular enzymes such as amylase and protease in several Bacillus species (S. T. Jørgensen, personal communication). On the basis of these findings, together with results for Streptomyces species, [15][16][17]19,20) improvement of RNA polymerase by the introduction of a rifampicin-resistance mutation might be a useful approach to realize bacterial capability.…”

“…However, unlike the case of NTD production, the S487L mutation failed to activate another secondary metabolism, bacilysin production. 46) Furthermore, in Streptomyces coelicolor and Streptomyces lividans, alteration at His-437 (corresponding to His-482 in B. subtilis) to Arg or Tyr effectively activated antibiotic production, 15,16,21) whereas these mutations were entirely ineffective in B. subtilis NTD production. This suggests that each rifampicin-resistance mutation has distinct effects in a gene-or organism-specific manner.…”

“…[10][11][12][13][14] In addition to these ribosomal mutations, rifampicin-resistance mutations in the rpoB gene encoding the -subunit of RNA polymelase were also found to activate antibiotic production in various microorganisms. [15][16][17][18][19][20][21] Thus, the targets of ribosome engineering have been expanded to RNA polymerase.…”