Genome concentration limits cell growth and modulates proteome composition in Escherichia coli

  1. Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA
  2. Sarafan Chemistry, Engineering, and Medicine for Human Health Institute, Stanford University, Stanford, CA 94305, USA
  3. Institute of Biotechnology, Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland
  4. Department of Biology, Stanford University, Stanford, CA 94305, USA
  5. Chan Zuckerberg Biohub, Stanford, CA 94305, USA
  6. Department of Genetics, Stanford University, Stanford, CA 94305, USA

Peer review process

Not revised: This Reviewed Preprint includes the authors’ original preprint (without revision), an eLife assessment, public reviews, and a provisional response from the authors.

Read more about eLife’s peer review process.

Editors

  • Reviewing Editor
    Ariel Amir
    Weizmann Institute of Science, Rehovot, Israel
  • Senior Editor
    Aleksandra Walczak
    École Normale Supérieure - PSL, Paris, France

Reviewer #1 (Public Review):

Summary:

The manuscript by Mäkelä et al. presents compelling experimental evidence that the amount of chromosomal DNA can become limiting for the total rate of mRNA transcription and consequently protein production in the model bacterium Escherichia coli. Specifically, the authors demonstrate that upon inhibition of DNA replication the single-cell growth rate continuously decreases, in direct proportion to the concentration of active ribosomes, as measured indirectly by single-particle tracking. The decrease of ribosomal activity with filamentation, in turn, is likely caused by a decrease of the concentration of mRNAs, as suggested by an observed plateau of the total number of active RNA polymerases. These observations are compatible with the hypothesis that DNA limits the total rate of transcription and thus translation. The authors also demonstrate that the decrease of RNAp activity is independent of two candidate stress response pathways, the SOS stress response and the stringent response, as well as an anti-sigma factor previously implicated in variations of RNAp activity upon variations of nutrient sources.

Remarkably, the reduction of growth rate is observed soon after the inhibition of DNA replication, suggesting that the amount of DNA in wild-type cells is tuned to provide just as much substrate for RNA polymerase as needed to saturate most ribosomes with mRNAs. While previous studies of bacterial growth have most often focused on ribosomes and metabolic proteins, this study provides important evidence that chromosomal DNA has a previously underestimated important and potentially rate-limiting role for growth.

Strengths:

This article links the growth of single cells to the amount of DNA, the number of active ribosomes and to the number of RNA polymerases, combining quantitative experiments with theory. The correlations observed during depletion of DNA, notably in M9gluCAA medium, are compelling and point towards a limiting role of DNA for transcription and subsequently for protein production soon after reduction of the amount of DNA in the cell. The article also contains a theoretical model of transcription-translation that contains a Michaelis-Menten type dependency of transcription on DNA availability and is fit to the data. While the model fits well with the continuous reduction of relative growth rate in rich medium (M9gluCAA), the behavior in minimal media without casamino acids is a bit less clear (see comments below).

At a technical level, single-cell growth experiments and single-particle tracking experiments are well described, suggesting that different diffusive states of molecules represent different states of RNAp/ribosome activities, which reflect the reduction of growth. However, I still have a few points about the interpretation of the data and the measured fractions of active ribosomes (see below).

Apart from correlations in DNA-deplete cells, the article also investigates the role of candidate stress response pathways for reduced transcription, demonstrating that neither the SOS nor the stringent response are responsible for the reduced rate of growth. Equally, the anti-sigma factor Rsd recently described for its role in controlling RNA polymerase activity in nutrient-poor growth media, seems also not involved according to mass-spec data. While other (unknown) pathways might still be involved in reducing the number of active RNA polymerases, the proposed hypothesis of the DNA substrate itself being limiting for the total rate of transcription is appealing.

Finally, the authors confirm the reduction of growth in the distant Caulobacter crescentus, which lacks overlapping rounds of replication and could thus have shown a different dependency on DNA concentration.

Weaknesses:

There are a range of points that should be clarified or addressed, either by additional experiments/analyses or by explanations or clear disclaimers.

First, the continuous reduction of growth rate upon arrest of DNA replication initiation observed in rich growth medium (M9gluCAA) is not equally observed in poor media. Instead, the relative growth rate is immediately/quickly reduced by about 10-20% and then maintained for long times, as if the arrest of replication initiation had an immediate effect but would then not lead to saturation of the DNA substrate. In particular, the long plateau of a constant relative growth rate in M9ala is difficult to reconcile with the model fit in Fig 4S2. Is it possible that DNA is not limiting in poor media (at least not for the cell sizes studied here) while replication arrest still elicits a reduction of growth rate in a different way? Might this have something to do with the naturally much higher oscillations of DNA concentration in minimal medium?

The authors argue that DNA becomes limiting in the range of physiological cell sizes, in particular for M9glCAA (Fig. 1BC). It would be helpful to know by how much (fold-change) the DNA concentration is reduced below wild-type (or multi-N) levels at t=0 in Fig 1B and how DNA concentration decays with time or cell area, to get a sense by how many-fold DNA is essentially 'overexpressed/overprovided' in wild-type cells.

Fig. 2: The distribution of diffusion coefficients of RpsB is fit to Gaussians on the log scale. Is this based on a model or on previous work or simply an empirical fit to the data? An exact analytical model for the distribution of diffusion constants can be found in the tool anaDDA by Vink, ..., Hohlbein Biophys J 2020. Alternatively, distributions of displacements are expressed analytically in other tools (e.g., in SpotOn).

The estimated fraction of active ribosomes in wild-type cells shows a very strong reduction with decreasing growth rate (down from 75% to 30%), twice as strong as measured in bulk experiments (Dai et al Nat Microbiology 2016; decrease from 90% to 60% for the same growth rate range) and probably incompatible with measurements of growth rate, ribosome concentrations, and almost constant translation elongation rate in this regime of growth rates. Might the different diffusive fractions of RpsB not represent active/inactive ribosomes? See also the problem of quantification above. The authors should explain and compare their results to previous work.

To measure the reduction of mRNA transcripts in the cell, the authors rely on the fluorescent dye SYTO RNAselect. They argue that 70% of the dye signal represents mRNA. The argument is based on the previously observed reduction of the total signal by 70% upon treatment with rifampicin, an RNA polymerase inhibitor (Bakshi et al 2014). The idea here is presumably that mRNA should undergo rapid degradation upon rif treatment while rRNA or tRNA are stable. However, work from Hamouche et al. RNA (2021) 27:946 demonstrates that rifampicin treatment also leads to a rapid degradation of rRNA. Furthermore, the timescale of fluorescent-signal decay in the paper by Bakshi et al. (half life about 10min) is not compatible with the previously reported rapid decay of mRNA (2-4min) but rather compatible with the slower, still somewhat rapid, decay of rRNA reported by Hamouche et al.. A bulk method to measure total mRNA as in the cited Balakrishnan et al. (Science 2022) would thus be a preferred method to quantify mRNA. Alternatively, the authors could also test whether the mass contribution of total RNA remains constant, which would suggest that rRNA decay does not contribute to signal loss. However, since rRNA dominates total RNA, this measurement requires high accuracy. The authors might thus tone down their conclusions on mRNA concentration changes while still highlighting the compelling data on RNAp diffusion.

The proteomics experiments are a great addition to the single-cell studies, and the correlations between distance from ori and protein abundance is compelling. However, I was missing a different test, the authors might have already done but not put in the manuscript: If DNA is indeed limiting the initiation of transcription, genes that are already highly transcribed in non-perturbed conditions might saturate fastest upon replication inhibition, while genes rarely transcribed should have no problem to accommodate additional RNA polymerases. One might thus want to test, whether the (unperturbed) transcription initiation rate is a predictor of changes in protein composition. This is just a suggestion the authors may also ignore, but since it is an easy analysis, I chose to mention it here.

Related to the proteomics, in l. 380 the authors write that the reduced expression close to the ori might reflect a gene-dosage compensatory mechanism. I don't understand this argument. Can the authors add a sentence to explain their hypothesis?

In Fig. 1E the authors show evidence that growth rate increases with cell length/area. While this is not a main point of the paper it might be cited by others in the future. There are two possible artifacts that could influence this experiment: a) segmentation: an overestimation of the physical length of the cell based on phase-contrast images (e.g., 200 nm would cause a 10% error in the relative rate of 2 um cells, but not of longer cells). b) time-dependent changes of growth rate, e.g., due to change from liquid to solid or other perturbations. To test for the latter, one could measure growth rate as a function of time, restricting the analysis to short or long cells, or measuring growth rate for short/long cells at selected time points. For the former, I recommend comparison of phase-contrast segmentation with FM4-64-stained cell boundaries.

Reviewer #2 (Public Review):

In this work, the authors uncovered the effects of DNA dilution on E. coli, including a decrease in growth rate and a significant change in proteome composition. The authors demonstrated that the decline in growth rate is due to the reduction of active ribosomes and active RNA polymerases because of the limited DNA copy numbers. They further showed that the change in the DNA-to-volume ratio leads to concentration changes in almost 60% of proteins, and these changes mainly stem from the change in the mRNA levels.

Reviewer #3 (Public Review):

Summary:

Mäkelä et al. here investigate genome concentration as a limiting factor on growth. Previous work has identified key roles for transcription (RNA polymerase) and translation (ribosomes) as limiting factors on growth, which enable an exponential increase in cell mass. While a potential limiting role of genome concentration under certain conditions has been explored theoretically, Mäkelä et al. here present direct evidence that when replication is inhibited, genome concentration emerges as a limiting factor.

Strengths:

A major strength of this paper is the diligent and compelling combination of experiment and modeling used to address this core question. The use of origin- and ftsZ-targeted CRISPRi is a very nice approach that enables dissection of the specific effects of limiting genome dosage in the context of a growing cytoplasm. While it might be expected that genome concentration eventually becomes a limiting factor, what is surprising and novel here is that this happens very rapidly, with growth transitioning even for cells within the normal length distribution for E. coli. Fundamentally, it demonstrates the fine balance of bacterial physiology, where the concentration of the genome itself (at least under rapid growth conditions) is no higher than it needs to be.

Weaknesses:

One limitation of the study is that genome concentration is largely treated as a single commodity. While this facilitates their modeling approach, one would expect that the growth phenotypes observed arise due to copy number limitation in a relatively small number of rate-limiting genes. The authors do report shifts in the composition of both the proteome and the transcriptome in response to replication inhibition, but while they report a positional effect of distance from the replication origin (reflecting loss of high-copy, origin-proximal genes), other factors shaping compositional shifts and their functional effects on growth are not extensively explored. This is particularly true for ribosomal RNA itself, which the authors assume to grow proportionately with protein. More generally, understanding which genes exert the greatest copy number-dependent influence on growth may aid both efforts to enhance (biotechnology) and inhibit (infection) bacterial growth.

Overall, this study provides a fundamental contribution to bacterial physiology by illuminating the relationship between DNA, mRNA, and protein in determining growth rate. While coarse-grained, the work invites exciting questions about how the composition of major cellular components is fine-tuned to a cell's needs and which specific gene products mediate this connection. This work has implications not only for biotechnology, as the authors discuss, but potentially also for our understanding of how DNA-targeted antibiotics limit bacterial growth.

Author response:

eLife assessment

This study represents a fundamental contribution to our understanding of how gene expression levels are controlled in bacteria. Through a series of compelling and careful experiments, relying on a mutant that blocks DNA replication but permits growth, and using various methods, the authors reveal how genome concentration rapidly becomes limiting for growth when replication is inhibited. This work contributes to our understanding of the contributions and limiting roles of DNA, mRNA, and ribosomes for growth in bacteria, and will be of considerable interest within both systems biology and microbial physiology.

Thank you!

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The manuscript by Mäkelä et al. presents compelling experimental evidence that the amount of chromosomal DNA can become limiting for the total rate of mRNA transcription and consequently protein production in the model bacterium Escherichia coli. Specifically, the authors demonstrate that upon inhibition of DNA replication the single-cell growth rate continuously decreases, in direct proportion to the concentration of active ribosomes, as measured indirectly by single-particle tracking. The decrease of ribosomal activity with filamentation, in turn, is likely caused by a decrease of the concentration of mRNAs, as suggested by an observed plateau of the total number of active RNA polymerases. These observations are compatible with the hypothesis that DNA limits the total rate of transcription and thus translation. The authors also demonstrate that the decrease of RNAp activity is independent of two candidate stress response pathways, the SOS stress response and the stringent response, as well as an anti-sigma factor previously implicated in variations of RNAp activity upon variations of nutrient sources.

Remarkably, the reduction of growth rate is observed soon after the inhibition of DNA replication, suggesting that the amount of DNA in wild-type cells is tuned to provide just as much substrate for RNA polymerase as needed to saturate most ribosomes with mRNAs. While previous studies of bacterial growth have most often focused on ribosomes and metabolic proteins, this study provides important evidence that chromosomal DNA has a previously underestimated important and potentially rate-limiting role for growth.

Thank you for the excellent summary of our work.

Strengths:

This article links the growth of single cells to the amount of DNA, the number of active ribosomes and to the number of RNA polymerases, combining quantitative experiments with theory. The correlations observed during depletion of DNA, notably in M9gluCAA medium, are compelling and point towards a limiting role of DNA for transcription and subsequently for protein production soon after reduction of the amount of DNA in the cell. The article also contains a theoretical model of transcription-translation that contains a Michaelis-Menten type dependency of transcription on DNA availability and is fit to the data. While the model fits well with the continuous reduction of relative growth rate in rich medium (M9gluCAA), the behavior in minimal media without casamino acids is a bit less clear (see comments below).

At a technical level, single-cell growth experiments and single-particle tracking experiments are well described, suggesting that different diffusive states of molecules represent different states of RNAp/ribosome activities, which reflect the reduction of growth. However, I still have a few points about the interpretation of the data and the measured fractions of active ribosomes (see below).

Apart from correlations in DNA-deplete cells, the article also investigates the role of candidate stress response pathways for reduced transcription, demonstrating that neither the SOS nor the stringent response are responsible for the reduced rate of growth. Equally, the anti-sigma factor Rsd recently described for its role in controlling RNA polymerase activity in nutrient-poor growth media, seems also not involved according to mass-spec data. While other (unknown) pathways might still be involved in reducing the number of active RNA polymerases, the proposed hypothesis of the DNA substrate itself being limiting for the total rate of transcription is appealing.

Finally, the authors confirm the reduction of growth in the distant Caulobacter crescentus, which lacks overlapping rounds of replication and could thus have shown a different dependency on DNA concentration.

Weaknesses:

There are a range of points that should be clarified or addressed, either by additional experiments/analyses or by explanations or clear disclaimers.

First, the continuous reduction of growth rate upon arrest of DNA replication initiation observed in rich growth medium (M9gluCAA) is not equally observed in poor media. Instead, the relative growth rate is immediately/quickly reduced by about 10-20% and then maintained for long times, as if the arrest of replication initiation had an immediate effect but would then not lead to saturation of the DNA substrate. In particular, the long plateau of a constant relative growth rate in M9ala is difficult to reconcile with the model fit in Fig 4S2. Is it possible that DNA is not limiting in poor media (at least not for the cell sizes studied here) while replication arrest still elicits a reduction of growth rate in a different way? Might this have something to do with the naturally much higher oscillations of DNA concentration in minimal medium?

We note that the total RNAP activity (abundance x active fraction) was also significantly reduced in poor media (Figure 3 -- supplement 4G and H) similarly to rich medium (Figure 3H). This is consistent with DNA being limiting. The main difference between rich and poor medium conditions is that the total ribosome activity in poor media (Figure 2 -- supplement 4G and H) was less affected in comparison to rich media (Figure 2H). Our interpretation of these results is that while DNA is limiting in all medium conditions (as shown by the RNAP data), changes in ribosome activity or mRNA degradation can compensate for the reduction in transcription in poor media and hence maintain better scaling of growth rates under DNA limitation. We understand how our current presentation made it confusing. We will reorganize the text and figures to better explain our results and interpretations.

The authors argue that DNA becomes limiting in the range of physiological cell sizes, in particular for M9glCAA (Fig. 1BC). It would be helpful to know by how much (fold-change) the DNA concentration is reduced below wild-type (or multi-N) levels at t=0 in Fig 1B and how DNA concentration decays with time or cell area, to get a sense by how many-fold DNA is essentially 'overexpressed/overprovided' in wild-type cells.

We will provide an estimate.

Fig. 2: The distribution of diffusion coefficients of RpsB is fit to Gaussians on the log scale. Is this based on a model or on previous work or simply an empirical fit to the data? An exact analytical model for the distribution of diffusion constants can be found in the tool anaDDA by Vink, ..., Hohlbein Biophys J 2020. Alternatively, distributions of displacements are expressed analytically in other tools (e.g., in SpotOn).

We use an empirical fit of Gaussian mixture model (GMM) of three states to the data and extract the fractions of molecules in each state. This avoids making too many assumptions on the underlying processes, e.g. a Markovian system with Brownian diffusion. The model in anaDDA (Vink et al.) is currently limited to two-transitioning states with a maximal step number of 8 steps per track for a computationally efficient solution (longer tracks are truncated). Using a short subset of the trajectories is less accurate than using the entire trajectory and because of this, we consider full tracks with at least 9 displacements. Meanwhile, Spot-On supports a three-state model but it is still based on a semi-analytical model with a pre-calculated library of parameters created by fitting of simulated data. Neither of these models considers the effect of cell confinement, which plays a major role on single-molecule diffusion in small-sized cells such as bacteria. For these reasons, we opted to use an empirical fit to the data. We note that the fractions of active ribosomes in WT cells grown in different media, which we extracted from these diffusion measurements, are consistent with estimates obtained by others using similar or different approaches (Forchhammer and Lindhal 1971; Mohapatra and Weisshaar, 2018; Sanamrad et al., 2014).

The estimated fraction of active ribosomes in wild-type cells shows a very strong reduction with decreasing growth rate (down from 75% to 30%), twice as strong as measured in bulk experiments (Dai et al Nat Microbiology 2016; decrease from 90% to 60% for the same growth rate range) and probably incompatible with measurements of growth rate, ribosome concentrations, and almost constant translation elongation rate in this regime of growth rates. Might the different diffusive fractions of RpsB not represent active/inactive ribosomes? See also the problem of quantification above. The authors should explain and compare their results to previous work.

We agree that our measured range is somewhat larger than the estimated range from Dai et al, 2016. However, they use different media, strains, and growth conditions. We also note that Dai et al did not make actual measurements of the active ribosome fraction. Instead, they calculate the “active ribosome equivalent” based on a model that includes growth rate, protein synthesis rate, RNA/protein abundance, and the total number of amino acids in all proteins in the cell. Importantly, our measurements show the same overall trend as Dai et al, 2016. Furthermore, our results are in quantitative agreements with previous experimental measurements that use ribosome profiling (Forchhammer and Lindhal 1971) or single-ribosome tracking (Mohapatra and Weisshaar, 2018; Sanamrad et al., 2014), which, we believe, validates our approach. We will clarify this point in the revised manuscript.

To measure the reduction of mRNA transcripts in the cell, the authors rely on the fluorescent dye SYTO RNAselect. They argue that 70% of the dye signal represents mRNA. The argument is based on the previously observed reduction of the total signal by 70% upon treatment with rifampicin, an RNA polymerase inhibitor (Bakshi et al 2014). The idea here is presumably that mRNA should undergo rapid degradation upon rif treatment while rRNA or tRNA are stable. However, work from Hamouche et al. RNA (2021) 27:946 demonstrates that rifampicin treatment also leads to a rapid degradation of rRNA. Furthermore, the timescale of fluorescent-signal decay in the paper by Bakshi et al. (half life about 10min) is not compatible with the previously reported rapid decay of mRNA (24min) but rather compatible with the slower, still somewhat rapid, decay of rRNA reported by Hamouche et al.. A bulk method to measure total mRNA as in the cited Balakrishnan et al. (Science 2022) would thus be a preferred method to quantify mRNA. Alternatively, the authors could also test whether the mass contribution of total RNA remains constant, which would suggest that rRNA decay does not contribute to signal loss. However, since rRNA dominates total RNA, this measurement requires high accuracy. The authors might thus tone down their conclusions on mRNA concentration changes while still highlighting the compelling data on RNAp diffusion.

Thank you for bringing the Hamouche et al 2022 paper to our attention. We will address this point in the revised manuscript.

The proteomics experiments are a great addition to the single-cell studies, and the correlations between distance from ori and protein abundance is compelling. However, I was missing a different test, the authors might have already done but not put in the manuscript: If DNA is indeed limiting the initiation of transcription, genes that are already highly transcribed in non-perturbed conditions might saturate fastest upon replication inhibition, while genes rarely transcribed should have no problem to accommodate additional RNA polymerases. One might thus want to test, whether the (unperturbed) transcription initiation rate is a predictor of changes in protein composition. This is just a suggestion the authors may also ignore, but since it is an easy analysis, I chose to mention it here.

Thank you for the suggestion. We will provide the suggested analysis in the revised manuscript.

Related to the proteomics, in l. 380 the authors write that the reduced expression close to the ori might reflect a gene-dosage compensatory mechanism. I don't understand this argument. Can the authors add a sentence to explain their hypothesis?

We apologize for the confusion. This will be addressed in the revised manuscript.

In Fig. 1E the authors show evidence that growth rate increases with cell length/area. While this is not a main point of the paper it might be cited by others in the future. There are two possible artifacts that could influence this experiment: a) segmentation: an overestimation of the physical length of the cell based on phase-contrast images (e.g., 200 nm would cause a 10% error in the relative rate of 2 um cells, but not of longer cells). b) time-dependent changes of growth rate, e.g., due to change from liquid to solid or other perturbations. To test for the latter, one could measure growth rate as a function of time, restricting the analysis to short or long cells, or measuring growth rate for short/long cells at selected time points. For the former, I recommend comparison of phasecontrast segmentation with FM4-64-stained cell boundaries.

As the reviewer notes, the small increase in relative growth was just a minor observation that does not affect our story whether it is biologically meaningful or the result of a technical artefact. But we agree with the reviewer that others might cite it in future works and thus should be interpreted with caution.

An artefact associated with time-dependent changes (e.g. changing from liquid cultures to more solid agarose pads) is unlikely for two reasons. 1. We show that varying the time that cells spend on agarose pads relative to liquid cultures does not affect the cell size-dependent growth rate results (Figure 1 -- supplement 5B). 2. We show that the growth rate is stable from the beginning of the time-lapse with no transient effects upon cell placement on agarose pads for imaging (Figure 1 -- supplement 5B). These results were described in the Methods section where they could easily be missed. We will revise the text to discuss these controls more prominently in the Results section.

As for cell segmentation, we have run simulations and agree with the reviewer that a small overestimation of cell area (which is possible with any cell segmentation methods including ours) could lead to a small increase in relative growth with increasing cell areas. Since the finding is not important to our story, we will simply alert the readers to the possibility that the observation may be due to a small cell segmentation bias.

Reviewer #2 (Public Review):

In this work, the authors uncovered the effects of DNA dilution on E. coli, including a decrease in growth rate and a significant change in proteome composition. The authors demonstrated that the decline in growth rate is due to the reduction of active ribosomes and active RNA polymerases because of the limited DNA copy numbers. They further showed that the change in the DNA-tovolume ratio leads to concentration changes in almost 60% of proteins, and these changes mainly stem from the change in the mRNA levels.

Thank you for the support and accurate summary!

Reviewer #3 (Public Review):

Summary:

Mäkelä et al. here investigate genome concentration as a limiting factor on growth. Previous work has identified key roles for transcription (RNA polymerase) and translation (ribosomes) as limiting factors on growth, which enable an exponential increase in cell mass. While a potential limiting role of genome concentration under certain conditions has been explored theoretically, Mäkelä et al. here present direct evidence that when replication is inhibited, genome concentration emerges as a limiting factor.

Strengths:

A major strength of this paper is the diligent and compelling combination of experiment and modeling used to address this core question. The use of origin- and ftsZ-targeted CRISPRi is a very nice approach that enables dissection of the specific effects of limiting genome dosage in the context of a growing cytoplasm. While it might be expected that genome concentration eventually becomes a limiting factor, what is surprising and novel here is that this happens very rapidly, with growth transitioning even for cells within the normal length distribution for E. coli. Fundamentally, it demonstrates the fine balance of bacterial physiology, where the concentration of the genome itself (at least under rapid growth conditions) is no higher than it needs to be.

Weaknesses:

One limitation of the study is that genome concentration is largely treated as a single commodity. While this facilitates their modeling approach, one would expect that the growth phenotypes observed arise due to copy number limitation in a relatively small number of rate-limiting genes. The authors do report shifts in the composition of both the proteome and the transcriptome in response to replication inhibition, but while they report a positional effect of distance from the replication origin (reflecting loss of high-copy, origin-proximal genes), other factors shaping compositional shifts and their functional effects on growth are not extensively explored. This is particularly true for ribosomal RNA itself, which the authors assume to grow proportionately with protein. More generally, understanding which genes exert the greatest copy number-dependent influence on growth may aid both efforts to enhance (biotechnology) and inhibit (infection) bacterial growth.

We agree but feel that identifying the specific limiting genes is beyond the scope of the study. However, to examine other potential contributing factors and identify limiting gene candidates, we plan to carry out new correlation analyses between our proteomic/transcriptomic datasets and published genome-wide datasets that report various variables under unperturbed conditions (e.g., mRNA/protein concentration, mRNA degradation rates, fitness cost, transcription/translation initiation rates, and essentiality).

Overall, this study provides a fundamental contribution to bacterial physiology by illuminating the relationship between DNA, mRNA, and protein in determining growth rate. While coarse-grained, the work invites exciting questions about how the composition of major cellular components is fine-tuned to a cell's needs and which specific gene products mediate this connection. This work has implications not only for biotechnology, as the authors discuss, but potentially also for our understanding of how DNA-targeted antibiotics limit bacterial growth.

Good point about the DNA-targeted antibiotics. Thank you!

  1. Howard Hughes Medical Institute
  2. Wellcome Trust
  3. Max-Planck-Gesellschaft
  4. Knut and Alice Wallenberg Foundation