Specific inhibition of bacterial RNase T2 by helix 41 of 16S ribosomal RNA

Abstract

Ribonuclease (RNase) T2 is involved in scavenging exogenous RNAs in the periplasmic space of bacteria. In Escherichia coli, although the 30S ribosomal subunit has long been known as a specific inhibitor of RNase T2 (designated as RNase I in E. coli), both the biochemical mechanisms and physiological roles of this interaction remain to be elucidated. Here we show, by creating hybrid ribosomes and mutational studies, that helix 41 (h41) of the E. coli 16S ribosomal RNA has a crucial role in the specific inhibition of RNase I. Notably, h41-mutant strains exhibit a lower survival rate at stationary phase and severe cell lysis when the post-segregation killing protein SrnB is expressed. These phenotypic defects accompany significant RNA degradation caused by RNase I. Thus, h41 in 16S rRNA provides a physiological benefit for the host cells in coping with the potential cytotoxicity of RNase T2.

Mutational robustness of 16S ribosomal RNA, shown by experimental horizontal gene transfer in Escherichia coli

“…Surprisingly, the 16S rRNA from not only the Gammaproteobacteria species Serratia ficaria (94.6% identity) but also from the evolutionarily distant Betaproteobacteria species Ralstonia pickettii (81.6% identity) have been found to form ribosomes active enough to support the growth of E. coli Δ7 (14). All these studies strongly suggest that the ribosome may be much more plastic than had previously been believed, perhaps enough to accommodate foreign 16S rRNA from diverse organisms.…”

“…Briefly, E. coli strain KT101 lacking all seven rRNA operons (rrnA, B, C, D, E, G, H) in its chromosome (14,17) was used as the host; its growth was complemented by the rrnB operon encoded in the rescue plasmid pRB101 (pSC101 ori, ampicillin resistant). The plasmid contains the counter selectable marker sacB and thus can be conditionally eliminated on addition of sucrose into the medium (14,17). Using general universal primers (18), we amplified approximately the entire length of the 16S rRNA gene (8-1,541, E. coli numbering) from environmental DNA.…”

“…This approach takes advantage of the large sequence (and the secondary structure's) diversity of the 16S rRNA genes in nature (16) and selects those that are functional in E. coli. Genetic selection was carried out as described previously (14) (Fig. S1).…”

“…The plasmid pKK3535 (E. coli rrnB, ampicillin resistant, pBR322 ori) in the SQ171 strain was replaced by pRB101 (E. coli rrnB, sacB, ampicillin resistant, pSC101 ori) to generate strain KT101 (14,17). KT101 was maintained at 37°C in 2× YT [1.6% (wt/vol) peptone, 1% (wt/vol) yeast extract, and 0.5% (wt/vol) NaCl] medium supplemented with 50 μg/mL ampicillin (14,17). Metagenome samples.…”

“…Recently, using experimental horizontal gene transfer (i.e., the interspecies exchange of E. coli 16S rRNA genes with a foreign counterpart), we have also shown that the active hybrid ribosome could be reconstituted in vivo by using E. coli Δ7, a null mutant of the ribosomal RNA (rrn) operon, as a host (14). Surprisingly, the 16S rRNA from not only the Gammaproteobacteria species Serratia ficaria (94.6% identity) but also from the evolutionarily distant Betaproteobacteria species Ralstonia pickettii (81.6% identity) have been found to form ribosomes active enough to support the growth of E. coli Δ7 (14).…”