A suitable streptomycin-resistant mutant for constructing unmarked in-frame gene deletions using rpsL as a counter-selection marker.

Yu-Kuo Tsai,Chang-Phone Fung,Ci-Hong Liou,Feng-Yee Chang,Ling Ma,L Kristopher Siu,Jung-Chung Lin,Christophe Herman

doi:10.1371/journal.pone.0109258

Abstract

The streptomycin counter-selection system is a useful tool for constructing unmarked in-frame gene deletions, which is a fundamental approach to study bacteria and their pathogenicity at the molecular level. A prerequisite for this system is acquiring a streptomycin-resistant strain due to rpsL mutations, which encodes the ribosomal protein S12. However, in this study no streptomycin resistance was found to be caused by rpsL mutations in all 127 clinical strains of Klebsiella pneumoniae isolated from liver abscess patients. By screening 107 spontaneous mutants of streptomycin resistance from a clinical strain of K. pneumoniae, nucleotide substitution or insertion located within the rpsL was detected in each of these strains. Thirteen different mutants with varied S12 proteins were obtained, including nine streptomycin-dependent mutants. The virulence of all four streptomycin-resistant mutants was further evaluated. Compared with the parental strain, the K42N, K42T and K87R mutants showed a reduction in growth rate, and the K42N and K42T mutants became susceptible to normal human serum. In the mice LD50 (the bacterial dose that caused 50% death) assay, the K42N and K42T mutants were ∼1,000-fold less lethal (∼2×105 CFU) and the K87R mutant was ∼50-fold less lethal (∼1×104 CFU) than the parental strain (∼2×102 CFU). A K42R mutant showed non-observable effects on the above assays, while this mutant exhibited a small cost (P<0.01) in an in vitro growth competition experiment. In summary, most of the K. pneumoniae strains with streptomycin resistance caused by rpsL mutations are less virulent than their parental strain in the absence of streptomycin. The K42R mutant showed similar pathogenicity to its parental strain and should be one of the best choices when using rpsL as a counter-selection marker.

Highlights

Streptomycin, the first aminoglycoside antibiotic, was reported in 1944 [1] and can inhibit protein synthesis by binding to the 30S ribosomal subunit
Our results demonstrated that rpsL mutations were rare in K. pneumoniae that was isolated from liver abscess patients, and previous studies have showed that different protein expression levels could be caused by rpsL mutations [26,28]
Streptomycin Susceptibility and Resistance Genes The 127 K. pneumoniae strains obtained from the liver abscess patients in different geographic localities were investigated as described in the Materials and Methods

Summary

Introduction

Streptomycin, the first aminoglycoside antibiotic, was reported in 1944 [1] and can inhibit protein synthesis by binding to the 30S ribosomal subunit. The most common mechanisms contributing to streptomycin resistance are aminoglycoside-modifying enzymes and target gene mutations [2], while 16S rRNA methylases, which have recently emerged, confer high-level resistance to all clinically available aminoglycosides except streptomycin [3,4,5]. Streptomycin resistance caused by its target genes is mediated by mutations in 16S rRNA encoded by rrs or ribosomal protein S12 encoded by rpsL [8]. The mutations of the S12 protein have often been found to confer a high-level streptomycin resistance by preventing streptomycin binding and/ or conferring ribosomal hyperaccuracy [8,10,11]. Some mutations could cause a strongly hyperaccurate phenotype and lead to streptomycin dependence [11]. The streptomycin-dependent mutants need streptomycin to keep their survival because their ribosomal proteins require the binding of streptomycin for properly function during protein synthesis [11]

Methods

Results

Conclusion