Comparative RNA function analysis reveals high functional similarity between distantly related bacterial 16 S rRNAs

The 16 S rRNA sequence has long been used uncritically as a molecular clock to infer phylogenetic relationships among prokaryotes without fully elucidating the evolutionary changes that this molecule undergoes. In this study, we investigated the functional evolvability of 16 S rRNA, using comparative RNA function analyses between the 16 S rRNAs of Escherichia coli (Proteobacteria) and Acidobacteria (78% identity, 334 nucleotide differences) in the common genetic background of E. coli. While the growth phenotype of an E. coli mutant harboring the acidobacterial gene was disrupted significantly, it was restored almost completely following introduction of a 16 S rRNA sequence with a single base-pair variation in helix 44; the remaining 332 nucleotides were thus functionally similar to those of E. coli. Our results suggest that 16 S rRNAs share an inflexible cradle structure formed by ribosomal proteins and have evolved by accumulating species-specific yet functionally similar mutations. While this experimental evidence suggests the neutral evolvability of 16 S rRNA genes and hence satisfies the necessary requirements to use the sequence as a molecular clock, it also implies the promiscuous nature of the 16 S rRNA gene, i.e., the occurrence of horizontal gene transfer among bacteria.


3
After incubation at 50°C for 1 h, the reaction products (2 µL) were introduced into competent E. coli JM109 cells (100 µL) and grown on LB/Tmp agar plates by incubating at 37°C overnight. The colonies were combined and the plasmids were extracted to yield a library.
This plasmid library was then used to transform KT101. A functional screen based on the viability of the transformed KT101 was carried out as described previously 4 , in which the selecting agent was changed from Zeocin to Tmp (10 µg/mL).

Domain-based chimeragenesis
Domain-based chimeragenesis was carried out between the 16S rRNA genes of Acidobacteria and E. coli using pRB105 carrying 16S NS11 as a template. Each domain (5′  For domain deletion in pRB105 carrying 16S NS11 , pRB105 was amplified inversely using sets of the following primers: Bac1r and CntD+ for the 5′ domain deletion, 5D-and 3MjD+ for the central domain deletion, CntD-and 3MnD+ for the 3′major domain deletion, and 3MjDand 1542f for the 3′minor domain deletion. PCR was performed using KOD Neo DNA polymerase (Toyobo) with the following temperature cycles: 94°C for 2 min, followed by 35 cycles of incubation at 94°C for 10 s, 48°C for 30 s, and 68°C for 15 s (for domain amplification) or 8 min (for domain deletion), and a final incubation at 68°C for 10 min. The amplicons were gel purified and dissolved in 30 µL of water. Each 16S rRNA domain fragment (~200 ng) and the cognate linearized vector fragment (~200 ng) were combined and ligated using the In-Fusion Cloning Kit (Clontech) in a total volume of 10 µL. After incubation at 50°C for 1 h, the products (2 µL) were introduced into competent E. coli JM109 cells. Correct shuffling products were confirmed by DNA sequencing and used for further studies.

In vitro translational activity
The KT105 derivative strains were grown in 600 mL of LB/Km/Tmp in a 3 L flask at 37°C to OD 600 0.5-0.6. Cells were pelleted by centrifugation (5,000 g, 10 min, 4°C), resuspended in 10 mL of cold RBS-H buffer (20 mM HEPES-KOH [pH 7.6], 10 mM Mg(OAc) 2 , 30 mM NH 4 Cl, 6 mM 2-mercaptethanol), and transferred into 2 mL tubes (1.2 mL each). The tubes were centrifuged (36,220 g, 10 min, 4°C), supernatant was discarded, and the pellets were 4 combined with equal amount of glass beads (YGB05, Yasui Kikai) and 500 µL of RBS-H buffer. Cells were disrupted in a Taitec bead crusher (µT-12) at maximal speed for 1 min at room temperature, followed by chilling in an ice water bath for 2 min. This cycle was repeated three times to ensure complete disruption of the cells. The mixture was centrifuged at 15,000 rpm (36,220 g) for 10 min at 4°C. The supernatant was then transferred to a new tube.
The cell extract was then layered on top of a 10-40% (w/v) sucrose gradient prepared in

Sucrose density gradient analysis
Sucrose density gradient analysis of the ribosomes was carried out as previously described 1 .
Briefly, KT105 derivatives were grown in 50 mL of LB/Km/Tmp in a 500 mL flask at 37°C.
When the OD 600 reached 0.4 to 0.6, 100 µg/mL chloramphenicol was added 5 min before harvesting to avoid polysome run off. The flask was rapidly chilled in ice water for 10 min, and the cells were collected by centrifugation (5,000 g, 10 min, 4°C). The pellets were Samples were taken from the top of the tube using a BIOCOMP Piston Gradient Fractionator, and A 254 was monitored continuously on an ATTO UV monitor (AC-5200). subunit (S1 is not included in the crystal structure). In the RNA-protein contact map (A), 88 of the total 410 RNA-protein interactions involved variable nucleotides. Thus, 73.7% nucleotides varied in NS11 do not interact with ribosomal proteins. Nucleotides 1416 and 1484 are not involved in interaction with proteins. It is known that genes for S9 and S17 can be knocked out in E. coli 18 , suggesting that interactions between 16S rRNA and these proteins are also not essential. The other 18 ribosomal protein genes are reported to be essential 18 . In the RNA-RNA contact map (B), the interactions of the compensatory nucleotides within the RNA helices were seen as patterns that protruded perpendicular to the diagonal line (the nucleotide interaction between 1416 and 1484 is also seen in a protrusion).
In this map, 285 nucleotides, representing 85.3% of the total 334 variable nucleotides, occurred in this protruding pattern, confirming the compensatory and conservative nature of the RNA secondary structure.