The ΔybeX strain accumulates distinct rRNA species already during the late exponential growth
As there is neither substantial assembly nor degradation of mature ribosomes in the stationary phase (Piir et al. , 2011), we conjectured that the accumulated fragments observed in ΔybeXcells were likely getting there by the late exponential phase. Accordingly, we purified ribosomal subunits from late exponential cells by sucrose gradient fractionation and assayed the rRNA composition of the 70S ribosomes, 50S and 30S ribosomal subunits by Northern blotting.
The sucrose gradient profiles for wild-type (WT) and ΔybeXlysates are very similar, with the vast majority of ribosomal particles being in the 70S ribosome fraction and the relatively minor free subunit fractions exhibiting no apparent abnormalities (Fig. 6A ). Northern blots revealed full-length 17S pre-rRNA in the 30S fractions of both the WT and the ΔybeX strains, as detected via 16S and 17S rRNA specific oligonucleotides (Fig. 6B -E ). In theΔybeX strain, the mature 16S rRNA is substantially reduced in the 30S fraction compared to the WT (Fig. 6B , C ). Thus, in the ΔybeX cells, the 30S (SSU) fraction was unlikely to contain many functionally active small ribosomal subunits.
In addition, ΔybeX cells accumulated distinct truncated 16S rRNA and 17S pre-rRNA fragments, missing the 3‘-end or 5‘-end, in the ribosomal fractions (Fig. 6B -E, denoted as “trunc.”). Firstly, a 5‘-end truncated 16S rRNA fragment is present in all ribosomal fractions of the ΔybeX strain, including the 70S ribosomes, as detected via 16S rRNA and 16S 3‘-end specific oligonucleotides (Fig. 6B , C ). Secondly, truncated 17S rRNA fragments were present only in the 30S fraction of ΔybeX, as detected via 17S 5‘ and 3‘ ends rRNA-specific probes (Fig. 6D ,E ). Truncated 17S pre-rRNA fragments, presumably arising from the precursor SSU particles, are absent in the 70S and 50S fractions (Fig. 6D , E ). Decay intermediates in the 30S fraction indicate that most pre-SSU particles are inactive and degradation-bound in late-exponential phase ΔybeX cultures. In contrast, the 23S rRNA specific probe reveals only relatively minor differences in degradation patterns between WT and ΔybeX strains (Fig. 6F ).
We tested the effect of chloramphenicol (CAM), a well-studied protein synthesis inhibitor (Wilson, 2014), treatment on the ribosomes by sucrose gradient fractionation and northern blotting (Fig. S5a ). The sucrose gradient profiles of the WT and ΔybeX strain lysates were similar, while CAM-treated ribosomal particles sedimented notably differently from those of mature subunits (Fig. S5b ; see also (Siibak et al. , 2009)). However, in the ΔybeXstrain, in Northern blots, we observed distinct aberrant 16S rRNA fragments, which were absent in the WT (Fig. S5c ). In addition, the CAM treatment, which leads to bacterial growth arrest, stabilizes the ΔybeX 30S particles. While CAM 30S particles contain a good measure of 16S rRNA and 17S pre-rRNA in the ΔybeX strain, theΔybeX 30S particles from the control experiment without CAM treatment contain fragments of SSU rRNA and reduced amounts of mature SSU rRNA (Fig. S5c ). These findings indicate that the accumulation of decay intermediates in ΔybeX is not an artefact that occurs during the purification of ribosomal samples but occursin vivo . This interpretation is further supported by the fact that hot phenol-extracted total RNA samples harbour similar fragments of rRNA (Fig. 5 ).
These results indicate that in the late exponential phase, RNA in most of the free 30S subunits of the ΔybeX strain is being fragmented. Moreover, the degradation fragments captured by the 16S rRNA and 17S rRNA-specific probes strongly suggest that in the ΔybeX strain, pre-ribosomes (in the 30S fraction) and mature ribosomes (in the 70S fraction) are susceptible to degradation.