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Antimicrobial Agents and Chemotherapy, October 2005, p. 4327-4334, Vol. 49, No. 10
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.10.4327-4334.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

High-Level Chromosomally Mediated Tetracycline Resistance in Neisseria gonorrhoeae Results from a Point Mutation in the rpsJ Gene Encoding Ribosomal Protein S10 in Combination with the mtrR and penB Resistance Determinants

Mei Hu,1,{dagger} Sobhan Nandi,1 Christopher Davies,2 and Robert A. Nicholas1*

Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7365,1 Department of Biochemistry, Medical University of South Carolina, Charleston, South Carolina2

Received 31 March 2005/ Returned for modification 21 April 2005/ Accepted 5 June 1005

Neisseria gonorrhoeae becomes resistant to tetracycline by two major mechanisms: expression of a plasmid-encoded TetM protein and mutations in endogenous genes (chromosomally mediated resistance). Early studies by Sparling and colleagues (P. F. Sparling F. A. J. Sarubbi, and E. Blackman, J. Bacteriol. 124:740-749, 1975) demonstrated that three genes were involved in high-level chromosomally mediated tetracycline resistance (MIC of tetracycline ≥ 2 µg/ml): ery-2 (now referred to as mtrR), penB, and tet-2. While the identities of the first two genes are known, the tet-2 gene has not been identified. We cloned the tet-2 gene, which confers tetracycline resistance, from tetracycline-resistant clinical isolate N. gonorrhoeae FA6140 and show that resistance is due to a single point mutation (Val-57 to Met) in the rpsJ gene (rpsJ1) encoding ribosomal protein S10. Moreover, the identical mutation was found in six distinct tetracycline-resistant clinical isolates in which the MIC of tetracycline was ≥2 µg/ml. Site-saturation mutagenesis of the codon for Val-57 identified two other amino acids (Leu and Gln) that conferred identical levels of resistance as the Met-57 mutation. The mutation maps to the vertex of a loop in S10 that is near the aminoacyl-tRNA site in the structure of the 30S ribosomal subunit from Thermus thermophilus, and the residue equivalent to Val-57 in T. thermophilus S10, Lys-55, is within 8 to 9 Å of bound tetracycline. These data suggest that large noncharged amino acids alter the rRNA structure near the tetracycline-binding site, leading to a lower affinity of the antibiotic.

* Corresponding author. Mailing address: Department of Pharmacology, University of North Carolina at Chapel Hill, CB#7365 Mary Ellen Jones Bldg., Chapel Hill, NC 27599-7365. Phone: (919) 966-6547. Fax: (919) 966-5640. E-mail: nicholas{at}med.unc.edu.

{dagger} Present address: Serenex, Inc., Durham, N.C.

Antimicrobial Agents and Chemotherapy, October 2005, p. 4327-4334, Vol. 49, No. 10
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.10.4327-4334.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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