Methylation of 23S rRNA nucleotide G745 is a secondary function of the RlmAI methyltransferase

Abstract

Several groups of Gram-negative bacteria possess an RlmAI methyltransferase that methylates 23S rRNA nucleotide G745 at the N1 position. Inactivation of rlmAI in Acinetobacter calcoaceticus and Escherichia coli reduces growth rates by at least 30%, supposedly due to ribosome malfunction. Wild-type phenotypes are restored by introduction of plasmid-encoded rlmAI, but not by the orthologous Gram-positive gene rlmAII that methylates the neighboring nucleotide G748. Nucleotide G745 interacts with A752 in a manner that does not involve the guanine N1 position. When a cytosine is substituted at A752, a Watson-Crick G745-C752 pair is formed occluding the guanine N1 and greatly reducing RlmAI methylation. Methylation is completely abolished by substitution of the G745 base. Intriguingly, the absence of methylation in E. coli rRNA mutant strains causes no reduction in growth rate. Furthermore, the slow-growing rlmA I knockout strains of Acinetobacter and E. coli revert to the wild-type growth phenotype after serial passages on agar plates. All the cells tested were pseudorevertants, and none of them had recovered G745 methylation. Analyses of the pseudorevertants failed to reveal second-site mutations in the ribosomal components close to nucleotide G745. The results indicate that cell growth is not dependent on G745 methylation, and that the RlmAI methyltransferase therefore has another (as yet unidentified) primary function.