Characterization of sparsomycin resistance in Streptomyces sparsogenes

Abstract

The antitumor antibiotic sparsomycin, produced by Streptomyces sparsogenes, is a universal translation inhibitor that blocks the peptide bond formation in ribosomes from all species. Sparsomycin-resistant strains were selected by transforming the sensitive Streptomyces lividans with an S. sparsogenes library. Resistance was linked to the presence of a plasmid containing an S. sparsogenes 5.9-kbp DNA insert. A restriction analysis of the insert traced down the resistance to a 3.6-kbp DNA fragment, which was sequenced. The analysis of the fragment nucleotide sequence together with the previous restriction data associate the resistance to srd, an open reading frame of 1,800 nucleotides. Ribosomes from S. sparsogenes and the S. lividans-resistant strains are equally sensitive to the inhibitor and bind the drug with similar affinity. Moreover, the drug was not modified by the resistant strains. However, resistant cells accumulated less antibiotic than the sensitive ones. In addition, membrane fractions from the resistant strains showed a higher capacity for binding the drug. The results indicate that resistance in the producer strain is not connected to either ribosome modification or drug inactivation, but it might be related to an alteration in the sparsomycin permeability barrier.

FIG. 1.

Susceptibility of S. lividans SLT4 to 90 μg (disk 1) and 60 μg (disk 2) of sparsomycin after (A) and before (B) the curing of plasmid pIJ702 containing the S. sparsogenes DNA insert.

FIG. 2.

Effect of antibiotics on the growth of S. lividans parental 3131 and SLT4 transformant strains. (A) E, erythromycin (60 μg); C, chloramphenicol (50 μg); S, sparsomycin (90 μg). (B) T, tetracycline (30 μg); L, lincomycin (60 μg); P, puromycin (150 μg). The antibiotics were placed on filter paper disks.

FIG. 3.

Subcloning of the S. sparsogenes DNA insert. Different fragments of the original insert in the plasmids isolated from S. lividans SLT4 were subcloned on pJI702 as indicated and tested for their capacity to induce sparsomycin resistance in wild-type S. lividans 1326.

FIG. 4.

Analysis of the nucleotide sequence of the 3.9-kbp fragment. Analysis of G+C content identified three putative ORFs in the sequence, marked srd, moxR, and tmp. The region included in the different inserts in Fig. 3 is indicated at the bottom. A vertical line marks the starting point of the S. sparsogenes DNA.

FIG. 5.

Nucleotide and deduced amino acid sequences of the srd ORF.

FIG. 6.

Sparsomycin inhibition of polyphenylalanine synthesis in cell extracts from S. sparsogenes (□), wild-type S. lividans (○), and S. lividans SLT4 (▵). Polymerizing activity was tested according to standard methods in the presence of the indicated concentrations of sparsomycin. Activity is shown as the percentage of the samples tested in the absence of drug.

FIG. 7.

Binding of ¹²⁵I-labeled phenol-sparsomycin to ribosomes from resistant and sensitive strains. Ribosomes (50 pmol) from S. sparsogenes (□) and S. lividans 3131 grown in the absence (○) or in the presence (▵) of 0.5 mg of thiostrepton/ml were incubated with increasing amounts of 0.1 μM ¹²⁵ I-labeled phenol-sparsomycin (10³ cpm/pmol), and the amount of bound antibiotic was estimated by filtration.

FIG. 8.

Accumulation of radioactive sparsomycin by sensitive and resistant Streptomyces cells. Cells from S. sparsogenes (○), S. lividans SLT4 (•), and S. lividans 3131 (□) grown to mid-exponential phase were collected, washed, and resuspended in binding buffer to an A₅₅₀ of 5.0. A total of 100,000 cpm of ¹²⁵I-labeled phenol-sparsomycin was added and the samples were incubated at 30°C. Cells were afterwards filtered through glass fiber filters and washed with binding buffer, and the radioactivity was estimated by scintillation counting.