Highlights d Bacteria produce just enough aaRSs to support the amino acid fluxes for translation d tRNA charging is not maximized at growth-optimized levels of aaRS production d Native levels of uncharged tRNAs have limited impacts on cell fitness d Stringent response alleviates fitness defects of aaRS underproduction in rich media
During steady-state cell growth, individual enzymatic fluxes can be directly inferred from growth rate by mass conservation, but the inverse problem remains unsolved. Perturbing the flux and expression of a single enzyme could have pleiotropic effects that may or may not dominate the impact on cell fitness. Here, we quantitatively dissect the molecular and global responses to varied expression of translation termination factors (peptide release factors, RFs) in the bacterium Bacillus subtilis. While endogenous RF expression maximizes proliferation, deviations in expression lead to unexpected distal regulatory responses that dictate fitness reduction. Molecularly, RF depletion causes expression imbalance at specific operons, which activates master regulators and detrimentally overrides the transcriptome. Through these spurious connections, RF abundances are thus entrenched by focal points within the regulatory network, in one case located at a single stop codon. Such regulatory entrenchment suggests that predictive bottom-up models of expression-fitness landscapes will require near-exhaustive characterization of parts.
Expression of motility genes is a potentially beneficial but costly process in bacteria. Interestingly, many isolate strains of Escherichia coli possess motility genes but have lost the ability to activate them under conditions in which motility is advantageous, raising the question of how they respond to these situations. Through transcriptome profiling of strains in the E. coli single-gene knockout Keio collection, we noticed drastic upregulation of motility genes in many of the deletion strains compared to levels in their weakly motile parent strain (BW25113). We show that this switch to a motile phenotype is not a direct consequence of the genes deleted but is instead due to a variety of secondary mutations that increase the expression of the major motility regulator, FlhDC. Importantly, we find that this switch can be reproduced by growing poorly motile E. coli strains in nonshaking liquid medium overnight but not in shaking liquid medium. Individual isolates after the nonshaking overnight incubations acquired distinct mutations upstream of the flhDC operon, including different insertion sequence (IS) elements and, to a lesser extent, point mutations. The rapidity with which genetic changes sweep through the populations grown without shaking shows that poorly motile strains can quickly adapt to a motile lifestyle by genetic rewiring. IMPORTANCE The ability to tune gene expression in times of need outside preordained regulatory networks is an essential evolutionary process that allows organisms to survive and compete. Here, we show that upon overnight incubation in liquid medium without shaking, populations of largely nonmotile Escherichia coli bacteria can rapidly accumulate mutants that have constitutive motility. This effect contributes to widespread secondary mutations in the single-gene knockout library, the Keio collection. As a result, 49/71 (69%) of the Keio strains tested exhibited various degrees of motility, whereas their parental strain is poorly motile. These observations highlight the plasticity of gene expression even in the absence of preexisting regulatory programs and should raise awareness of procedures for handling laboratory strains of E. coli.
During steady-state cell growth, individual enzymatic fluxes can be directly inferred from growth rate by mass conservation, but the inverse problem remains unsolved. Perturbing the flux and expression of a single enzyme could have pleiotropic effects that may or may not dominate the impact on cell fitness. Here we quantitatively dissect the molecular and global responses to varied expression of translation termination factors (peptide release factors, RFs) in bacterium Bacillus subtilis. While endogenous RF expression maximizes proliferation, deviations in expression lead to unexpected distal regulatory responses that dictate fitness reduction. Molecularly, RF depletion causes expression imbalance at specific operons, which activates master regulators and detrimentally overrides the transcriptome. Through these spurious connections, RF abundances are thus entrenched by focal points within the regulatory network, in one case located at a single stop codon. Such regulatory entrenchment suggests that predictive bottom-up models of expression-fitness landscapes will require near-exhaustive characterization of parts.HighlightsPrecision measurements enable multiscale expression-to-fitness mapping.RF depletion leads to imbalanced translation for co-transcribed gene pairs.Imbalanced translation induces unintended regulons to the detriment of cell fitness.Swapping a single stop codon rewires global susceptibility to RF perturbation.
In many bacteria, ribosomal proteins autogenously repress their own expression by interacting with RNA structures typically located in the 5′-UTRs of their mRNA transcripts. This regulation is necessary to maintain a balance between ribosomal proteins and rRNA to ensure proper ribosome production. Despite advances in noncoding RNA discovery and validation of RNA-protein regulatory interactions, the selective pressures that govern the formation and maintenance of such RNA cis-regulators in the context of an organism remain largely undetermined. To examine the impact disruptions to this regulation have on bacterial fitness, we introduced point mutations that abolish ribosomal protein binding and regulation into the RNA structure that controls expression of ribosomal proteins L20 and L35 within the Bacillus subtilis genome. Our studies indicate that removing this regulation results in reduced log phase growth, improper rRNA maturation, and the accumulation of a kinetically trapped or misassembled ribosomal particle at low temperatures, suggesting defects in ribosome synthesis. Such work emphasizes the important role regulatory RNAs play in the stoichiometric production of ribosomal components for proper ribosome composition and overall organism viability and reinforces the potential of targeting ribosomal protein production and ribosome assembly with novel antimicrobials.
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