Global protein turnover quantification in Escherichia coli reveals cytoplasmic recycling under nitrogen limitation.

Protein turnover is critical for proteostasis, but turnover quantification is challenging, and even in well-studied E. coli, proteome-wide measurements remain scarce. Here, we quantify the turnover rates of ~3200 E. coli proteins under 13 conditions by combining heavy isotope labeling with complement reporter ion quantification and find that cytoplasmic proteins are recycled when nitrogen is limited. We use knockout experiments to assign substrates to the known cytoplasmic ATP-dependent proteases. Surprisingly, none of these proteases are responsible for the observed cytoplasmic protein degradation in nitrogen limitation, suggesting that a major proteolysis pathway in E. coli remains to be discovered. Lastly, we show that protein degradation rates are generally independent of cell division rates. Thus, we present broadly applicable technology for protein turnover measurements and provide a rich resource for protein half-lives and protease substrates in E. coli, complementary to genomics data, that will allow researchers to study the control of proteostasis.

Global and gene-specific translational regulation in Escherichia coli across different conditions.

How well mRNA transcript levels represent protein abundances has been a controversial issue. Particularly across different environments, correlations between mRNA and protein exhibit remarkable variability from gene to gene. Translational regulation is likely to be one of the key factors contributing to mismatches between mRNA level and protein abundance in bacteria. Here, we quantified genome-wide transcriptome and relative translation efficiency (RTE) under 12 different conditions in Escherichia coli. By quantifying the mRNA-RTE correlation both across genes and across conditions, we uncovered a diversity of gene-specific translational regulations, cooperating with transcriptional regulations, in response to carbon (C), nitrogen (N), and phosphate (P) limitations. Intriguingly, we found that many genes regulating translation are themselves subject to translational regulation, suggesting possible feedbacks. Furthermore, a random forest model suggests that codon usage partially predicts a gene's cross-condition variability in translation efficiency; such cross-condition variability tends to be an inherent quality of a gene, independent of the specific nutrient limitations. These findings broaden the understanding of translational regulation under different environments and provide novel strategies for the control of translation in synthetic biology. In addition, our data offers a resource for future multi-omics studies.

Steric interactions and out-of-equilibrium processes control the internal organization of bacteria.

Despite the absence of a membrane-enclosed nucleus, the bacterial DNA is typically condensed into a compact body-the nucleoid. This compaction influences the localization and dynamics of many cellular processes including transcription, translation, and cell division. Here, we develop a model that takes into account steric interactions among the components of the Escherichia coli transcriptional-translational machinery (TTM) and out-of-equilibrium effects of messenger RNA (mRNA) transcription, translation, and degradation, to explain many observed features of the nucleoid. We show that steric effects, due to the different molecular shapes of the TTM components, are sufficient to drive equilibrium phase separation of the DNA, explaining the formation and size of the nucleoid. In addition, we show that the observed positioning of the nucleoid at midcell is due to the out-of-equilibrium process of mRNA synthesis and degradation: mRNAs apply a pressure on both sides of the nucleoid, localizing it to midcell. We demonstrate that, as the cell grows, the production of these mRNAs is responsible for the nucleoid splitting into two lobes and for their well-known positioning to 1/4 and 3/4 positions on the long cell axis. Finally, our model quantitatively accounts for the observed expansion of the nucleoid when the pool of cytoplasmic mRNAs is depleted. Overall, our study suggests that steric interactions and out-of-equilibrium effects of the TTM are key drivers of the internal spatial organization of bacterial cells.

Monitoring mammalian mitochondrial translation with MitoRiboSeq.

Several essential components of the electron transport chain, the major producer of ATP in mammalian cells, are encoded in the mitochondrial genome. These 13 proteins are translated within mitochondria by 'mitoribosomes'. Defective mitochondrial translation underlies multiple inborn errors of metabolism and has been implicated in pathologies such as aging, metabolic syndrome and cancer. Here, we provide a detailed ribosome profiling protocol optimized to interrogate mitochondrial translation in mammalian cells (MitoRiboSeq), wherein mitoribosome footprints are generated with micrococcal nuclease and mitoribosomes are separated from cytosolic ribosomes and other RNAs by ultracentrifugation in a single straightforward step. We highlight critical steps during library preparation and provide a step-by-step guide to data analysis accompanied by open-source bioinformatic code. Our method outputs mitoribosome footprints at single-codon resolution. Codons with high footprint densities are sites of mitoribosome stalling. We recently applied this approach to demonstrate that defects in mitochondrial serine catabolism or in mitochondrial tRNA methylation cause stalling of mitoribosomes at specific codons. Our method can be applied to study basic mitochondrial biology or to characterize abnormalities in mitochondrial translation in patients with mitochondrial disorders.

Modeling microbial metabolic trade-offs in a chemostat.

Microbes face intense competition in the natural world, and so need to wisely allocate their resources to multiple functions, in particular to metabolism. Understanding competition among metabolic strategies that are subject to trade-offs is therefore crucial for deeper insight into the competition, cooperation, and community assembly of microorganisms. In this work, we evaluate competing metabolic strategies within an ecological context by considering not only how the environment influences cell growth, but also how microbes shape their chemical environment. Utilizing chemostat-based resource-competition models, we exhibit a set of intuitive and general procedures for assessing metabolic strategies. Using this framework, we are able to relate and unify multiple metabolic models, and to demonstrate how the fitness landscape of strategies becomes intrinsically dynamic due to species-environment feedback. Such dynamic fitness landscapes produce rich behaviors, and prove to be crucial for ecological and evolutionarily stable coexistence in all the models we examined.

A Dual-Mechanism Antibiotic Kills Gram-Negative Bacteria and Avoids Drug Resistance.

The rise of antibiotic resistance and declining discovery of new antibiotics has created a global health crisis. Of particular concern, no new antibiotic classes have been approved for treating Gram-negative pathogens in decades. Here, we characterize a compound, SCH-79797, that kills both Gram-negative and Gram-positive bacteria through a unique dual-targeting mechanism of action (MoA) with undetectably low resistance frequencies. To characterize its MoA, we combined quantitative imaging, proteomic, genetic, metabolomic, and cell-based assays. This pipeline demonstrates that SCH-79797 has two independent cellular targets, folate metabolism and bacterial membrane integrity, and outperforms combination treatments in killing methicillin-resistant Staphylococcus aureus (MRSA) persisters. Building on the molecular core of SCH-79797, we developed a derivative, Irresistin-16, with increased potency and showed its efficacy against Neisseria gonorrhoeae in a mouse vaginal infection model. This promising antibiotic lead suggests that combining multiple MoAs onto a single chemical scaffold may be an underappreciated approach to targeting challenging bacterial pathogens.

Near-equilibrium glycolysis supports metabolic homeostasis and energy yield.

Glycolysis plays a central role in producing ATP and biomass. Its control principles, however, remain incompletely understood. Here, we develop a method that combines 2H and 13C tracers to determine glycolytic thermodynamics. Using this method, we show that, in conditions and organisms with relatively slow fluxes, multiple steps in glycolysis are near to equilibrium, reflecting spare enzyme capacity. In Escherichia coli, nitrogen or phosphorus upshift rapidly increases the thermodynamic driving force, deploying the spare enzyme capacity to increase flux. Similarly, respiration inhibition in mammalian cells rapidly increases both glycolytic flux and the thermodynamic driving force. The thermodynamic shift allows flux to increase with only small metabolite concentration changes. Finally, we find that the cellulose-degrading anaerobe Clostridium cellulolyticum exhibits slow, near-equilibrium glycolysis due to the use of pyrophosphate rather than ATP for fructose-bisphosphate production, resulting in enhanced per-glucose ATP yield. Thus, near-equilibrium steps of glycolysis promote both rapid flux adaptation and energy efficiency.

Escherichia coli translation strategies differ across carbon, nitrogen and phosphorus limitation conditions.

For cells to grow faster they must increase their protein production rate. Microorganisms have traditionally been thought to accomplish this increase by producing more ribosomes to enhance protein synthesis capacity, leading to the linear relationship between ribosome level and growth rate observed under most growth conditions previously examined. Past studies have suggested that this linear relationship represents an optimal resource allocation strategy for each growth rate, independent of any specific nutrient state. Here we investigate protein production strategies in continuous cultures limited for carbon, nitrogen and phosphorus, which differentially impact substrate supply for protein versus nucleic acid metabolism. Unexpectedly, we find that at slow growth rates, Escherichia coli achieves the same protein production rate using three different strategies under the three different nutrient limitations. Under phosphorus (P) limitation, translation is slow due to a particularly low abundance of ribosomes, which are RNA-rich and thus particularly costly for phosphorous-limited cells. Under nitrogen (N) limitation, translation elongation is slowed by processes including ribosome stalling at glutamine codons. Under carbon (C) limitation, translation is slowed by accumulation of inactive ribosomes not bound to messenger RNA. These extra ribosomes enable rapid growth acceleration during nutrient upshift. Thus, bacteria tune ribosome usage across different limiting nutrients to enable balanced nutrient-limited growth while also preparing for future nutrient upshifts.

Metabolic interactions between dynamic bacterial subpopulations.

Individual microbial species are known to occupy distinct metabolic niches within multi-species communities. However, it has remained largely unclear whether metabolic specialization can similarly occur within a clonal bacterial population. More specifically, it is not clear what functions such specialization could provide and how specialization could be coordinated dynamically. Here, we show that exponentially growing Bacillus subtilis cultures divide into distinct interacting metabolic subpopulations, including one population that produces acetate, and another population that differentially expresses metabolic genes for the production of acetoin, a pH-neutral storage molecule. These subpopulations exhibit distinct growth rates and dynamic interconversion between states. Furthermore, acetate concentration influences the relative sizes of the different subpopulations. These results show that clonal populations can use metabolic specialization to control the environment through a process of dynamic, environmentally-sensitive state-switching.

Mitochondrial translation requires folate-dependent tRNA methylation.

Folates enable the activation and transfer of one-carbon units for the biosynthesis of purines, thymidine and methionine. Antifolates are important immunosuppressive and anticancer agents. In proliferating lymphocytes and human cancers, mitochondrial folate enzymes are particularly strongly upregulated. This in part reflects the need for mitochondria to generate one-carbon units and export them to the cytosol for anabolic metabolism. The full range of uses of folate-bound one-carbon units in the mitochondrial compartment itself, however, has not been thoroughly explored. Here we show that loss of the catalytic activity of the mitochondrial folate enzyme serine hydroxymethyltransferase 2 (SHMT2), but not of other folate enzymes, leads to defective oxidative phosphorylation in human cells due to impaired mitochondrial translation. We find that SHMT2, presumably by generating mitochondrial 5,10-methylenetetrahydrofolate, provides methyl donors to produce the taurinomethyluridine base at the wobble position of select mitochondrial tRNAs. Mitochondrial ribosome profiling in SHMT2-knockout human cells reveals that the lack of this modified base causes defective translation, with preferential mitochondrial ribosome stalling at certain lysine (AAG) and leucine (UUG) codons. This results in the impaired expression of respiratory chain enzymes. Stalling at these specific codons also occurs in certain inborn errors of mitochondrial metabolism. Disruption of whole-cell folate metabolism, by either folate deficiency or antifolate treatment, also impairs the respiratory chain. In summary, mammalian mitochondria use folate-bound one-carbon units to methylate tRNA, and this modification is required for mitochondrial translation and thus oxidative phosphorylation.

Paracrine and autocrine signals induce and maintain mesenchymal and stem cell states in the breast.

The epithelial-mesenchymal transition (EMT) has been associated with the acquisition of motility, invasiveness, and self-renewal traits. During both normal development and tumor pathogenesis, this change in cell phenotype is induced by contextual signals that epithelial cells receive from their microenvironment. The signals that are responsible for inducing an EMT and maintaining the resulting cellular state have been unclear. We describe three signaling pathways, involving transforming growth factor (TGF)-ß and canonical and noncanonical Wnt signaling, that collaborate to induce activation of the EMT program and thereafter function in an autocrine fashion to maintain the resulting mesenchymal state. Downregulation of endogenously synthesized inhibitors of autocrine signals in epithelial cells enables the induction of the EMT program. Conversely, disruption of autocrine signaling by added inhibitors of these pathways inhibits migration and self-renewal in primary mammary epithelial cells and reduces tumorigenicity and metastasis by their transformed derivatives.