Impairment of ribosomal subunit synthesis in aminoglycoside-treated ribonuclease mutants of Escherichia coli

Abstract

The bacterial ribosome is an important target for many antimicrobial agents. Aminoglycoside antibiotics bind to both 30S and 50S ribosomal subunits, inhibiting translation and subunit formation. During ribosomal subunit biogenesis, ribonucleases (RNases) play an important role in rRNA processing. E. coli cells deficient for specific processing RNases are predicted to have an increased sensitivity to neomycin and paromomycin. Four RNase mutant strains showed an increased growth sensitivity to both aminoglycoside antibiotics. E. coli strains deficient for the rRNA processing enzymes RNase III, RNase E, RNase G or RNase PH showed significantly reduced subunit amounts after antibiotic treatment. A substantial increase in a 16S RNA precursor molecule was observed as well. Ribosomal RNA turnover was stimulated, and an enhancement of 16S and 23S rRNA fragmentation was detected in E. coli cells deficient for these enzymes. This work indicates that bacterial RNases may be novel antimicrobial targets.

Fig. 1

Bacterial rRNA processing pathways. The major rRNA processing enzymes are indicated. The binding of both aminoglycosides to 16S and 23S rRNAs is also shown (modified from (Davies et al. 2010). John Wiley and Sons used with permission.

Fig. 2

Sucrose gradient profiles of ³H uridine labeled ribosomal subunits isolated from E. coli cells grown with or without 10μg/mL antibiotics. Gradient profiles for wild type (a), RNase III deficient (b), RNase E deficient (c), RNase I/PH/R deficient (d), RNase G deficient (e), and RNase I/II/PNPase/PH deficient (f). Results are the means of 2 gradient profiles.

Fig. 3

Agilent gel analysis of total RNA from wild type and mutant cells grown with and without antibiotics. Control (C), neomycin (N), paromomycin (P)

Fig. 4

Northern blot analysis of 16S rRNA fragmentation for wild type and RNase mutant E. coli cells. RNA was isolated from cells grown without and with antibiotics. RNA sequences were identified by hybridization with a 16S DNA probe. Control (C), neomycin (N), paromomycin (P)

Fig. 5

Northern blot analysis of 23S rRNA fragmentation for wild type and RNase mutant E. coli cells. RNA was isolated from cells grown without and with antibiotics. RNA sequences were identified by hybridization with a 23S DNA probe. Control (C), neomycin (N), paromomycin (P)