The ∆ybeX strain accumulates distinct rRNA species already during the late exponential growth
As there is neither assembly nor degradation of mature ribosomes in the early stationary phase (Piir et al. , 2011), we conjectured that the 16S fragments observed in ∆ybeX cells were likely accumulating by the late exponential phase. Accordingly, we purified, from late exponential cells, ribosomal subunits by sucrose gradient fractionation and analyzed the rRNA composition of the 70S ribosomes, as well as 50S and 30S subunits by Northern blotting. In these experiments we used probes specific for both ends of 17S precursor, for the 16S 3’ end, and for the mature 16S rRNA and 23S rRNA, allowing us to see degradation intermediates emanating from immature pre-16S rRNAs (Fig. 6A ).
The sucrose gradient profiles for WT and ∆ybeX lysates are very similar, with the vast majority of ribosomal particles being in the presumably active 70S ribosome fraction and the small free subunit fractions exhibiting no obvious abnormalities (Fig. 6B ). The Northern blots revealed 17S precursor rRNAs in the 30S fractions of both the WT and the ∆ybeX strain, likely due to active ribosomal synthesis in both strains (Fig. 6C, E ). In addition, in the∆ybeX strain the mature 16S rRNA species is substantially reduced in the 30S fraction, so that the 17S to 16S ratio is clearly shifted in relation to WT. Thus, in the ∆ybeX strain the 30S fraction is unlikely to contain many functionally active ribosomal subunits.
In addition, there are two distinct 16S fragments, both around 1 kb long (truncated ribosomal RNA species denoted as “trunc.”), in the ribosomal fractions originating from the ∆ybeX cells. Firstly, there is a major 5’ end-truncated 16S rRNA fragment which is present in all ribosomal fractions, including the 70S ribosomes (Fig. 6C, E ). In the 30S fraction this fragment is produced already from the 17S pre-rRNA (Fig. 6F ), but the same fragment in the 70S ribosomes is not of this origin, presumably originating from full length mature 16S rRNA inside the 70S particles (Fig. 6E ). Its presence in the 30S fraction is more pronounced than that of the 17S pre-rRNA, indicating that most of the pre-30S particles are inactive and degradation-bound in late exponential phase ΔybeX cultures. Secondly, there is a slightly larger 3’ end-truncated 16S RNA fragment (Fig. 6E ), which is present in the 30S fraction only (Fig. 6G ). This fragment also originates from the 17S precursor particles. In contrast, 23S rRNA specific probe reveals several relatively minor differences in degradation patterns between WT andΔybeX strains (Fig. 6D ).
Taken together, these results indicate that in the late exponential phase the majority of free 30S ΔybeX strain is in the process of being degraded. Moreover, the degradation fragments captured by the pre-16S rRNA specific probes strongly suggest that in the ΔybeXstrain both pre-ribosomes (in the 30S fraction) and mature ribosomes (in the 70S fraction) are susceptible to degradation. While a majority of pre-ribosomes in the 30S fraction appear as the 1000-nt rRNA fragment (Fig. 6E, F ), a minority of mature 70S is present as the 1000-nt fragment.