Discovery of a small molecule that inhibits bacterial ribosome biogenesis

Thank you for sending your work entitled “Discovery of a small molecule that inhibits bacterial ribosome biogenesis” for consideration at eLife. Your article has been favorably evaluated by Michael Marletta (Senior editor), Roberto Kolter (Reviewing editor), and 2 reviewers.

The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

In this manuscript Stoke et al use a chemogenetic screen to discover a small molecule, lamotrigine (lam) that induces cold-sensitivity in E. coli. The authors then proceed to demonstrate that lam perturbs assembly of 30S and 50S ribosomes. Suppressor analysis and biochemical analysis demonstrate that the translation initiation factor IF2 is the target of lam. Surprisingly though, lam does not perturb translation.

This is an interesting and exciting paper with intriguing implications. However, it was felt that some minor additional experimental support would solidify the claims. In particular, growth curves and/or serial dilution plating of bacterial strains at the permissive (37°C), nonpermissive (15°C), and various temperatures in between using media (LB and M9) containing various concentrations of lamotrigene (including concentrations used in various experiments) should be presented. 15°C is very low temperature for growth of E. coli. The doubling time for the DMSO/wild-type control at 15°C is about 12x that at 37°C and this raises the question of how much cold shock plays into these data. It is unclear why such a low temperature was chosen for this study. Results from the proposed simple experiments would allow more confident correlation of defects in ribosome biogenesis with effects on cell growth.

In addition, there are several places where the presentation of the data is difficult to follow and thus the authors should modify the text in response to the minor comments listed below.

1) Presumably the cold sensitivity factor is the ratio of growth at 37°C, relative to 15°C, relative to wild type?

2) Error bars would be desirable in Figure 1B considering that the % cold sensitivity for the Co-enzyme metabolism gene class was only ∼2 fold less than the Translation, ribosome structure, and biogenesis gene class. This difference was the foundation for all the subsequent work.

3) Figure 4 is extremely difficult to follow: What is the difference in the experiment between 4B and C? It seems that panel B says that there is no measurable accumulation of pre-16S rRNA, but pre-23S rRNA is accumulated in the pre-30S and the pre-50S peak. Nevertheless, panel C says that pre-16S rRNA also accumulates. Where are these discrepancies coming from? Also, perhaps clearer labeling could help. I would call p16S, pre-16S; perhaps one could call +7 and +3, pre-pre50S, and pre-50S, respectively; or perhaps pre-50S+3 or pre-50S+7; it is also not explained in the legend where the +7 is coming from; it took me a while to figure that out.

4) What is Figure 4D supposed to show; what is on the x-axis? Neither the text nor the legend is helpful.

5) It is hard to tell if S3 is depleted from the pre-30S as claimed. I'd say no...

6) The effects on 50S maturation seem to be far more drastic than the effects on 30S maturation; perhaps that should be discussed.

7) It would be nice to show a structure of mature 30S and 50S illustrating which of the RPs are missing. Also, the authors point out that IF2 overexpression rescues the YjeQ deletion; interestingly the YjeQ deletion has a very similar phenotype in terms of depleted proteins.

8) The data indicates a maturation-specific function of the drug, and perhaps even of domain II. This should be discussed, and a domainII-specific function should be tested by complementation with IF2 from divergent species at low temperature.

Growth curves and/or serial dilution plating of bacterial strains at the permissive (37°C), nonpermissive (15°C), and various temperatures in between using media (LB and M9) containing various concentrations of lamotrigene (including concentrations used in various experiments) should be presented. 15°C is very low temperature for growth of E. coli. The doubling time for the DMSO/wild-type control at 15°C is about 12x that at 37^°C and this raises the question of how much cold shock plays into these data. It is unclear why such a low temperature was chosen for this study. Results from the proposed simple experiments would allow more confident correlation of defects in ribosome biogenesis with effects on cell growth.

These experiments have been completed, and the results can be found in Figure 2–figure supplement 2. We observed a media-independent phenotype where lamotrigine activity was inversely proportional to growth temperature. These results are consistent with the conclusion that lamotrigine inhibits a cold sensitive event during ribosome biogenesis. As temperature decreases, the apparent role of IF2 in ribosome biogenesis becomes increasingly essential, thus increasing the growth inhibitory effect of lamotrigine. 15°C was selected in this study to maximize cold-induced ribosome biogenesis phenotypes while still permitting cell growth amenable to high-throughput screening in micro-well format. Based on the new data shown in Figure 2–figure supplement 2, we believe that lamotrigine would produce identical ribosome biogenesis phenotypes at higher temperatures, albeit at correspondingly higher concentrations.

1) Presumably the cold sensitivity factor is the ratio of growth at 37°C, relative to 15°C, relative to wild type?

The cold sensitivity factor is defined in Figure legend 1 as the ratio of growth at 37°C to growth at 15°C, normalized to the mean of all cold sensitivity factors calculated. In response to the reviewers comment, we have also noted this normalization in the Results section.

2) Error bars would be desirable in Figure 1B considering that the % cold sensitivity for the Co-enzyme metabolism gene class was only ∼2 fold less than the Translation, ribosome structure, and biogenesis gene class. This difference was the foundation for all the subsequent work.

Figure 1B is a histogram showing the ratio of cold sensitive gene deletion strains in each COG functional class to the total number of non-essential E. coli genes in that same functional class. As such, calculating error to determine statistical significance of these results is not possible. Alternatively, we performed a permutation test to determine the probability of observing a 21% hit rate in the translation, ribosome structure & biogenesis functional class by chance. By permuting the classification assignments, we determined that the 21% cold sensitive genes in the Translation class was significant with a bootstrapped p-value < 1e^-6. This p-value has been added to the legend of Figure 1B.

3) Figure 4 is extremely difficult to follow: What is the difference in the experiment between 4B and C? It seems that panel B says that there is no measurable accumulation of pre-16S rRNA, but pre-23S rRNA is accumulated in the pre-30S and the pre-50S peak. Nevertheless, panel C says that pre-16S rRNA also accumulates. Where are these discrepancies coming from? Also, perhaps clearer labeling could help. I would call p16S, pre-16S; perhaps one could call +7 and +3, pre-pre50S, and pre-50S, respectively; or perhaps pre-50S+3 or pre-50S+7; it is also not explained in the legend where the +7 is coming from; it took me a while to figure that out.

This is indeed a complex figure and these comments have helped us to improve the clarity of these panels. We have modified Figure 4B, the legend for Figure 4B and have re-written the Results section describing the relationship between Figure 4B and 4C. Briefly, Figure 4B illustrates the proportion of processed rRNA in various positions through the gradient. This proportion of processed rRNA is defined as [immature rRNA/total rRNA] for each species analyzed (either 16S or 23S). This is ideal for understanding the rRNA processing efficiency of individual ribosomal particles. However, as was seen in the pre-30S region of the gradient in lamotrigine-treated cells, we were able to purify measurable quantities of both 16S and 23S species that were unprocessed. Thus, to discern whether the dominant species residing within the pre-30S region was 30S or 50S in nature, we needed to determine absolute quantities of 16S rRNA species and 23S rRNA species (Figure 4C) originating from this region. Performing this experiment with 5’ primer extension of both 16S and 23S rRNA in parallel from the same sucrose gradient fractions allowed us to conclude that the major species in the pre-30S peak was an immature 30S particle.

4) What is Figure 4D is supposed to show; what is on the x-axis? Neither the text nor the legend is helpful.

We have greatly expanded the figure legend and our discussion of this figure. We have also modified the legend for Figure 4–figure supplement 2 to avoid any additional confusion caused by figures of this form. This panel is intended to show example r-protein occupancy data of small and large subunit proteins (S3, S15, L28, L24) along sucrose gradients of DMSO- and lamotrigine-treated cells. Blue represents early fractions in the gradient and red indicates late fractions. The vertical axis quantifies r-protein occupancy. For each protein, the horizontal axis spans different fractions along the sucrose gradient from lowest density (blue; left) to highest density (red; right).

5) It is hard to tell if S3 is depleted from the pre-30S as claimed. I'd say no...

Although the depletion effect on S3 is less pronounced than that on S2 or S21, we contend that the protein is in fact depleted from the pre-30S peak in the lamotrigine treated cells and the 30S peak in the DMSO-treated cells. The effect is most apparent by comparing the intensity in fractions 1-4 of S3 with those from S8, S15, S6, and S18 in Figure 4E. Clearly the S3 abundance is lower than that of S8, etc. Additionally, the decreased abundance of S3 can be seen in the DMSO-treated cells by comparing the 30S peak intensities (fractions 7-9) of S3 to those of S20, S11, S12, etc. In response to the reviewers concerns, we have modified the Results section to emphasize the effect on S2 and S21 and to deemphasize the effect on S3. Critically, this change has no effect on the overall conclusions of the manuscript.

6) The effects on 50S maturation seem to be far more drastic than the effects on 30S maturation; perhaps that should be discussed.

We agree that the data demonstrate very strong effects of lamotrigine treatment on 50S assembly. However, as evidenced by the significant accumulation of immature 16S rRNA in the pre-30S fractions, the effects on 30S assembly are profound in their own right. In the absence of a detailed mechanism for IF2 function in ribosome biogenesis, we have chosen to give equal weight to the effects observed on the 30S and 50S subunits. We contend that apparent assembly defects may be more pronounced in the large subunit simply as a result of the size and complexity of this subunit relative to the 30S. As a result of this complexity, even mild assembly inhibition may lead to the accumulation of the discrete particle we observe. Indeed, such effects have been observed upon depletion of a variety of assembly factors, some of which are non-essential. For example, deletion of SrmB results in the accumulation of a discrete 40S particle despite relatively mild effects on cellular growth rate (Moore, 2012).

7) It would be nice to show a structure of mature 30S and 50S illustrating which of the RPs are missing. Also, the authors point out that IF2 overexpression rescues the YjeQ deletion; interestingly the YjeQ deletion has a very similar phenotype in terms of depleted proteins.

Images highlighting the depleted ribosomal proteins can be found in Figure 4–figure supplement 2 panels C, D. We have modified the figure legends for these panels to clarify their content. We have also added a discussion point hypothesizing the functional relationship between YjeQ and IF2 during cold stress in E. coli.

8) The data indicates a maturation-specific function of the drug, and perhaps even of domain II. This should be discussed, and a domainII-specific function should be tested by complementation with IF2 from divergent species at low temperature.

We postulated the potential role(s) of IF2 in ribosome biogenesis in the Discussion. However, we attempted to keep this section concise in order to avoid over-interpretation of our data. Further investigation of the role of domain II in ribosome biogenesis through complementation is a great idea, and has previously been indirectly conducted. Two thorough investigations by Laalami et al. (Maguire, 2009; Williamson, 2005) showed that truncations of the N-terminus of IF2 resulted in E. coli strains that were incapable of growing at temperatures below 40°C. These results are in agreement with the cold sensitive phenotype induced by lamotrigine, and strongly suggest that complementing E. coli IF2 with the infB gene product from divergent species lacking domain II would result in cells that similarly display cold sensitivity.