title: A suitable streptomycin-resistant mutant for constructing unmarked in-frame gene deletions using rpsL as a counter-selection marker.
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 × 10(5) CFU) and the K87R mutant was ∼ 50-fold less lethal (∼ 1 × 10(4) CFU) than the parental strain (∼ 2 × 10(2) 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.
authors.affiliation: Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Miaoli, Taiwan.
authors.forename: Yu-Kuo
authors.initials: YK
authors.lastname: Tsai
authors.affiliation: Division of Infectious Diseases and Tropical Medicine, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.
authors.forename: Ci-Hong
authors.initials: CH
authors.lastname: Liou
authors.affiliation: Division of Infectious Diseases and Tropical Medicine, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.
authors.forename: Jung-Chung
authors.initials: JC
authors.lastname: Lin
authors.affiliation: Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Miaoli, Taiwan.
authors.forename: Ling
authors.initials: L
authors.lastname: Ma
authors.affiliation: Section of Infectious Diseases, Department of Medicine, Taipei Veterans General Hospital and National Yang-Ming University, Taipei, Taiwan.
authors.forename: Chang-Phone
authors.initials: CP
authors.lastname: Fung
authors.affiliation: Division of Infectious Diseases and Tropical Medicine, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan; Taiwan Centres for Disease Control, Taipei, Taiwan.
authors.forename: Feng-Yee
authors.initials: FY
authors.lastname: Chang
authors.affiliation: Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Miaoli, Taiwan; Division of Infectious Diseases and Tropical Medicine, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan; Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan.
authors.forename: L Kristopher
authors.initials: LK
authors.lastname: Siu
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005819:Genetic Markers; D006801:Humans; D007710:Klebsiella Infections / Q000382:microbiology / Q000401:mortality / Q000473:pathology; D007711:Klebsiella pneumoniae / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D008100:Liver Abscess / Q000382:microbiology / Q000473:pathology; D051379:Mice; D008826:Microbial Sensitivity Tests; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins / Q000172:deficiency / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013307:Streptomycin / Q000494:pharmacology; D016019:Survival Analysis
chemical_list: D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; D005819:Genetic Markers; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins; C000717190:RpsI protein, E coli; C506766:RpsL protein, E coli; D013307:Streptomycin