The last rRNA methyltransferase of E. coli revealed: the yhiR gene encodes adenine-N6 methyltransferase specific for modification of A2030 of 23S ribosomal RNA

Abstract

The ribosomal RNA (rRNA) of Escherichia coli contains 24 methylated residues. A set of 22 methyltransferases responsible for modification of 23 residues has been described previously. Herein we report the identification of the yhiR gene as encoding the enzyme that modifies the 23S rRNA nucleotide A2030, the last methylated rRNA nucleotide whose modification enzyme was not known. YhiR prefers protein-free 23S rRNA to ribonucleoprotein particles containing only part of the 50S subunit proteins and does not methylate the assembled 50S subunit. We suggest renaming the yhiR gene to rlmJ according to the rRNA methyltransferase nomenclature. The phenotype of yhiR knockout gene is very mild under various growth conditions and at the stationary phase, except for a small growth advantage at anaerobic conditions. Only minor changes in the total E. coli proteome could be observed in a cell devoid of the 23S rRNA nucleotide A2030 methylation.

FIGURE 1.

Location of the methylated residue m⁶A2030 of the 23S rRNA in the 50S subunit of E. coli. Shown is a structure of the E. coli 50S ribosomal subunit as viewed from the 30S side. Modified nucleotide m⁶A2030 is shown as dark gray van der Waals sphere model. (Inset) A closer view of the interacting m⁶A2030 and U571 nucleotides.

FIGURE 2.

In vitro modification of potential YhiR substrates prepared from the ΔyhiR strain. Methylation of the 50S subunits, NH4Cl/ethanol split particles, LiCl split particles, and deproteinized 23S rRNA prepared from the ΔyhiR strain.

FIGURE 3.

MALDI MS analysis of the RNase A digest of the 23S rRNA fragment's 2017–2053 nt. The part of spectrum around mass/charge (m/z) 1990–2050 is shown. MALDI spectra correspond to fragments of the 23S from the (A) wild-type strain; (B) ΔyhiR strain; (C) ΔyhiR strain, transformed with pCA24YhiR; (D) 50S subunits from the ΔyhiR strain, treated with SAM and recombinant YhiR in vitro; and (E) deproteinized 23S rRNA from the ΔyhiR strain, treated with SAM and recombinant YhiR in vitro. Oligonucleotides corresponding to peaks are indicated.

FIGURE 4.

(A) Cell titer drop in the logarithmic scale after incubation of bacterial cultures over time. Data points and approximation curves for the wild-type strain are shown in black, while those for the ΔyhiR strain are shown in gray. Squares correspond to incubation in LB at 42°C. Stars correspond to incubation in LB at 37°C. Diamonds correspond to incubation in LB at 30°C. Circles correspond to incubation in M9 at 42°C. Crosses correspond to incubation in M9 at 37°C. Triangles correspond to incubation in M9 at 30°C. Additionally, incubation conditions are indicated on the right side of the figure at the ends of the corresponding approximation curves. (B) Growth of the wild-type strain (black curve) and the ΔyhiR strain (gray curve) at anaerobic conditions. Error bars, SDs from an average of five independently grown cultures.