∆ybeX perturbs ribosomal assembly through a separate mechanism from chloramphenicol
The strong sensitivity of ∆ybeX cells to chloramphenicol (CAM) treatment prompted us to investigate the chloramphenicol phenotype further. CAM is a well-studied inhibitor of protein synthesis that binds to the large ribosomal subunit, inhibiting peptidyl transfer (Wilson, 2014). The effect of chloramphenicol on cell growth is at least partially mediated by the imbalanced synthesis of r-proteins, which results in the accumulation of partially assembled and misassembled ribosomal subunits (Siibak et al. , 2009).
We tested the effect of sub-inhibitory concentrations of CAM on the ribosomes using sucrose gradient fractionation and northern blotting. Overnight-grown cells were diluted in liquid LB medium and grown until cells reached mid-exponential growth (OD600=0.3). The CAM treatment took place for 2 hours. The cells were also grown without CAM for 2 hours as a control (Fig. 7A ).
While the CAM particles were formed in both wild type and ∆ybeXstrains (Fig. 7B ), we failed to observe any aberrant rRNA species for the WT strain (Fig. 7C ), while the accumulation of the distinct rRNA species appeared in ∆ybeX cells repeatedly (Fig. 7D ). Thus, the mechanism that leads to the degradation of pre-rRNA in 30S particles in ∆ybeX cells seems to be different from that of the imbalanced protein synthesis caused by CAM. The CAM action mechanism also appears to stabilize ∆ybeX 30S particles, while the pre-16S rRNA degradation intermediate is present in both 70S and 50S fractions. We believe its presence in the 50S to be due to cross-contamination from the 70S fraction. Interestingly, while WT CAM 70S particles contain a good measure of 17S pre-rRNA (which is absent in WT non-treated cultures), the ∆ybeX CAM 70S particles, although containing the degradation intermediate, do not have this pre-16S rRNA species. These results suggest that the perturbation of assembly by CAM and by ∆ybeX go by separate and at least partially independent mechanisms.