Acquisition of Streptomycin Resistance by Oxidative Stress Induced by Hydrogen Peroxide in Radiation-Resistant Bacterium Deinococcus geothermalis

Abstract

Streptomycin is used primarily to treat bacterial infections, including brucellosis, plague, and tuberculosis. Streptomycin resistance easily develops in numerous bacteria through the inhibition of antibiotic transfer, the production of aminoglycoside-modifying enzymes, or mutations in ribosomal components with clinical doses of streptomycin treatment. (1) Background: A transposable insertion sequence is one of the mutation agents in bacterial genomes under oxidative stress. (2) Methods: In the radiation-resistant bacterium Deinococcus geothermalis subjected to chronic oxidative stress induced by 20 mM hydrogen peroxide, active transposition of an insertion sequence element and several point mutations in three streptomycin resistance (SmR)-related genes (rsmG, rpsL, and mthA) were identified. (3) Results: ISDge6 of the IS5 family integrated into the rsmG gene (dgeo_2335), called SrsmG, encodes a ribosomal guanosine methyltransferase resulting in streptomycin resistance. In the case of dgeo_2840-disrupted mutant strains (S1 and S2), growth inhibition under antibiotic-free conditions was recovered with increased growth yields in the presence of 50 µg/mL streptomycin due to a streptomycin-dependent (SmD) mutation. These mutants have a predicted proline-to-leucine substitution at the 91st residue of ribosomal protein S12 in the decoding center. (4) Conclusions: Our findings show that the active transposition of a unique IS element under oxidative stress conditions conferred antibiotic resistance through the disruption of rsmG. Furthermore, chronic oxidative stress induced by hydrogen peroxide also induced streptomycin resistance caused by point and frameshift mutations of streptomycin-interacting residues such as K43, K88, and P91 in RpsL and four genes for streptomycin resistance.

Keywords: Deinococcus geothermalis; chronic oxidative stress; insertion sequence; phenotypic variation; ribosomal protein S12 (RpsL); streptomycin-dependent (SmD) and -resistant (SmR); transposition.

Figure 1

The selection of streptomycin-resistant (SmR) and -dependent (SmD) clones by chronic oxidative stress of wild-type, Δdgeo_0257, Δdgeo_0281, and Δdgeo_2840 parent strains. (A) Growth comparison on TGY medium containing 50 µg/mL streptomycin among parent (P) and mutant (S1–S4 and rsmG) strains. The red box indicates an Sm-dependent phenotype. (B) Minimum inhibitory concentration (MIC) values using the disc diffusion method for all selected mutants within the range of 10 to 250 µg/mL streptomycin for parent strains and 10,000 to 100,000 µg/mL streptomycin for Sm-resistant mutant strains on TGY medium.

Figure 2

Comparison of growth profiles among Δdgeo_2840 parent and three Sm-resistant mutant strains (A) in antibiotic-free TGY medium and the SmD phenotype of Δdgeo_2840 S1 and S2. The arrow indicates the point at which 50 µg/mL streptomycin was added (B).

Figure 3

PCR detection of insertion sequence (IS) transposition in four Sm resistance-related genes in wild-type (WT), Δdgeo_0257, Δdgeo_0281, and Δdgeo_2840 mutants. Lanes: M, size marker; 1, WT; 2–5, WT S1–S4; 6, Δdgeo_0257 S1; 7–9, Δdgeo_2840 S1, S2, and SrsmG; and 10, Δdgeo_0281 S1. The arrow indicates a larger PCR product caused by IS integration in dgeo_2335 (rsmG).

Figure 4

Detection of insertion sequence (IS) transposition and loci of point mutations in the Sm resistance-related genes rsmG (dgeo_2335) (A), mthA (dgeo_0776) (B), and rpsL (dgeo_1873) (C). The ISDge6 element of the IS5 family was integrated at the 58th nucleotide of rsmG (A). Streptomycin resistance mutants from wild-type, Δdgeo_0257, Δdgeo_0281, and Δdgeo_2840 mutant strains have frameshift or point mutations in three Sm resistance-related genes. All mutations are also summarized in Table 1.

Figure 4

Detection of insertion sequence (IS) transposition and loci of point mutations in the Sm resistance-related genes rsmG (dgeo_2335) (A), mthA (dgeo_0776) (B), and rpsL (dgeo_1873) (C). The ISDge6 element of the IS5 family was integrated at the 58th nucleotide of rsmG (A). Streptomycin resistance mutants from wild-type, Δdgeo_0257, Δdgeo_0281, and Δdgeo_2840 mutant strains have frameshift or point mutations in three Sm resistance-related genes. All mutations are also summarized in Table 1.

Figure 5

Three-dimensional (3D) view of the decoding center area, including ribosomal proteins S12 and S4 and the three 16S rRNA helices h18, h27, and h44 from the 30S small ribosomal subunit topology of T. thermophilus HB8 with streptomycin from the Protein Data Bank (RCSB PDB, www.pdb.org; accession number 4DV7; original contributors: Demirci H, Murphy IVF, Murphy E, Gregory ST, Dahlberg AE, and Jogl G.) [29]. From the deposited structure data, the decoding region was “zoomed-in” using Mol* Viewer [28], and the streptomycin-interacting amino acid residues of ribosomal proteins (violet color) and nucleotides of 16S rRNA helices (green color) were marked.

Figure 6

qRT-PCR for Sm-enhanced clone growth of Δdgeo_2840 parent and two SmD strains (S1 and S2) with the wild type at OD₆₀₀ of 2.0 and 4.0. dgeo_0646 and dgeo_1869 are EF-Tu genes (tufA and tufB, respectively), and dgeo_1202 is a putative aadA gene. The statistics were performed with a probability t-test in the Prism program with p < 0.05 (*), p < 0.001 (**), and p < 0.0001 (***).