Mutations in rsmG, encoding a 16S rRNA methyltransferase, result in low-level streptomycin resistance and antibiotic overproduction in Streptomyces coelicolor A3(2)

Abstract

Certain str mutations that confer high- or low-level streptomycin resistance result in the overproduction of antibiotics by Streptomyces spp. The str mutations that confer the high-level resistance occur within rpsL, which encodes the ribosomal protein S12, while those that cause low-level resistance are not as well known. We have used comparative genome sequencing to determine that low-level resistance is caused by mutations of rsmG, which encodes an S-adenosylmethionine (SAM)-dependent 16S rRNA methyltransferase containing a SAM binding motif. Deletion of rsmG from wild-type Streptomyces coelicolor resulted in the acquisition of streptomycin resistance and the overproduction of the antibiotic actinorhodin. Introduction of wild-type rsmG into the deletion mutant completely abrogated the effects of the rsmG deletion, confirming that rsmG mutation underlies the observed phenotype. Consistent with earlier work using a spontaneous rsmG mutant, the strain carrying DeltarsmG exhibited increased SAM synthetase activity, which mediated the overproduction of antibiotic. Moreover, high-performance liquid chromatography analysis showed that the DeltarsmG mutant lacked a 7-methylguanosine modification in the 16S rRNA (possibly at position G518, which corresponds to G527 of Escherichia coli). Like certain rpsL mutants, the DeltarsmG mutant exhibited enhanced protein synthetic activity during the late growth phase. Unlike rpsL mutants, however, the DeltarsmG mutant showed neither greater stability of the 70S ribosomal complex nor increased expression of ribosome recycling factor, suggesting that the mechanism underlying increased protein synthesis differs in the rsmG and the rpsL mutants. Finally, spontaneous rsmG mutations arose at a 1,000-fold-higher frequency than rpsL mutations. These findings provide new insight into the role of rRNA modification in activating secondary metabolism in Streptomyces.

FIG. 1.

SignalMap (NimbleGen) representation of CGS analysis of the str-1 mutant strain KO-132. The lowest two traces show the signal intensities for the M145 wild-type (green) and str-1 mutant (blue) hybridizations; the red trace above shows their ratio. The top line depicts an SNP confirmed by sequencing.

FIG. 2.

Effect of deleting the rsmG gene on the level of Sm resistance. The 1147 (wild-type [WT]) and KO-656 (ΔrsmG) strains were grown for 2 days on GYM agar with (+SM) or without (−SM) 2 μg/ml Sm. Strain KO-179 (rsmG mutant [str-19]) served as a reference.

FIG. 3.

Effect of deleting the rsmG gene on Act production and expression of actII-ORF4. (A) Act production by S. coelicolor strains 1147 (wild-type [WT]) and KO-656 (ΔrsmG). Upper row shows the reverse side of the plates. Strains were incubated on an R4C agar plate for 4 days. (B) Act production by S. coelicolor strains 1147 and KO-656 in R5⁻ liquid medium. (C) Expression of actII-ORF4 mRNA by cells grown on R4C agar plates for the indicated times, as determined by RT-PCR. (D) Effect of rsmG mutation on Act and Red production by S. lividans grown on R4 agar plates for 6 days. The upper (left) and reverse (right) sides of the plates are shown. Blue and red represent actinorhodin (Act) and undecylprodigiosin (Red), respectively.

FIG. 4.

HPLC profile of 16S rRNA nucleosides from wild-type (upper panel) and ΔrsmG mutant (lower panel) strains. 16S rRNA was isolated, digested completely with nuclease P1 and alkaline phosphatase, and analyzed by HPLC. The peak position for m⁷G was determined using standard m⁷G.

FIG. 5.

Effect of deleting rsmG on SAM synthetase activity. (A) SAM synthetase activity in wild-type (1147) and ΔrsmG mutant (KO-656) strains. Cells were grown on R5⁻ agar covered with cellophane. Samples were taken at the indicated times, and SAM synthetase activity was determined. One unit of activity is defined as the amount of enzyme that changed the optical density at 340 nm at a rate of 12.4/min. (B) Expression of metK-xylE fusion element, as determined by quantitative catechol dioxygenase assays in wild-type and ΔrsmG mutant strains grown on R5⁻ agar medium. XylE activity was determined as described previously (25).

FIG. 6.

Profiles of growth and in vitro protein synthesis in wild-type 1147 (WT) and ΔrsmG (KO-656) strains. (A) Growth in YEME medium at 30°C was monitored by measuring the optical density at 450 nm (OD₄₅₀). The zero time point represents 22 to 24 h after inoculation of fresh spores, when the OD₄₅₀ was 0.2; “S2” indicates the early stationary phase (see reference 13). (B) In vitro synthesis of GFP using wild-type and ΔrsmG ribosomes prepared from cells grown to S₂ phase. Strain KO-178 (K88E rpsL mutant) served as a reference strain. Equal aliquots (10 μl) of reaction mixture were withdrawn at the indicated times and subjected to electrophoresis in 10% polyacrylamide gels. The intensity of the GFP bands was determined by scanning the fluorographs. (C) Effects on GFP synthesis of cross-mixing the S-150 fractions and ribosomes from wild-type and mutant cells grown in YEME medium to stationary (S₂) phase. Cell-free translation of GFP mRNA was performed as described in the panel B legend. (Upper panel) Fluorographs of synthesized GFP. (Lower panel) Relative levels of GFP synthesis. (D) Expression profile of RRF protein in wild-type (1147) and mutant (ΔrsmG) strains. Strain KO-178 (K88E rpsL mutant) was the reference strain.