When Too Much ATP Is Bad for Protein Synthesis

Pontes, Mauricio H.; Sevostyanova, Anastasia; Groisman, Eduardo A.

doi:10.1016/j.jmb.2015.06.021

Cited by 98 publications

(88 citation statements)

References 110 publications

(136 reference statements)

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“…Growth defects and magnitudes of the effects are not reflected here. thought to stabilize ribosomes (16). As such, magnesium deprivation can result in cessation of translation due to dissociation of ribosomes (16).…”

Section: Discussionmentioning

confidence: 99%

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Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media

Christensen

Orr

Rao

et al. 2017

Appl Environ Microbiol

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Complex media are routinely used to cultivate diverse bacteria. However, this complexity can obscure the factors that govern cell growth. While studying protein acetylation in buffered tryptone broth supplemented with glucose (TB7-glucose), we observed that Escherichia coli did not fully consume glucose prior to stationary phase. However, when we supplemented this medium with magnesium, the glucose was completely consumed during exponential growth, with concomitant increases in cell number and biomass but reduced cell size. Similar results were observed with other sugars and other peptide-based media, including lysogeny broth. Magnesium also limited cell growth for Vibrio fischeri and Bacillus subtilis in TB7-glucose. Finally, magnesium supplementation reduced protein acetylation. Based on these results, we conclude that growth in peptide-based media is magnesium limited. We further conclude that magnesium supplementation can be used to tune protein acetylation without genetic manipulation. These results have the potential to reduce potentially deleterious acetylated isoforms of recombinant proteins without negatively affecting cell growth.IMPORTANCE Bacteria are often grown in complex media. These media are thought to provide the nutrients necessary to grow bacteria to high cell densities. In this work, we found that peptide-based media containing a sugar are magnesium limited for bacterial growth. In particular, magnesium supplementation is necessary for the bacteria to use the sugar for cell growth. Interestingly, in the absence of magnesium supplementation, the bacteria still consume the sugar. However, rather than use it for cell growth, the bacteria instead use the sugar to acetylate lysines on proteins. As lysine acetylation may alter the activity of proteins, this work demonstrates how lysine acetylation can be tuned through magnesium supplementation. These findings may be useful for recombinant protein production, when acetylated isoforms are to be avoided. They also demonstrate how to increase bacterial growth in complex media.KEYWORDS acetylation, complex media, improved cell growth, magnesium, tryptone B acteria are routinely grown in complex media containing a rich assortment of nutrients. For example, lysogeny broth (LB) contains tryptone and yeast exact. Together, these components provide a rich mixture of nutrients that supports growth of numerous bacteria. However, the complexity of such media obscures factors governing cell behavior, especially those that limit cell growth.We routinely grow Escherichia coli aerobically in buffered tryptone broth (TB7) supplemented with 4 g/liter glucose (TB7-glucose) when studying protein acetylation.

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Section: Discussionmentioning

confidence: 99%

“…thought to stabilize ribosomes (16). As such, magnesium deprivation can result in cessation of translation due to dissociation of ribosomes (16). Under the magnesiumstarved conditions found in complex peptide-based media, it would not be surprising for ribosome control to be a mechanism by which cells cease growth and division.…”

Section: Discussionmentioning

confidence: 99%

Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media

Christensen

Orr

Rao

et al. 2017

Appl Environ Microbiol

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“…Intriguingly, both corL and mgtM use the inefficient start codon UUG, which could be especially sensitive to global changes in translation, such as ribosome availability 37 . Because the majority of cytosolic Mg 2+ is devoted to maintenance of ribosome structure and activity, an increase in Mg 2+ uptake may help the cell cope with translational stressors 38 . Therefore, Rho’s involvement in the control of Mg 2+ transporter genes may promote Mg 2+ uptake when mRNA translation is impaired.…”

Section: Discussionmentioning

confidence: 99%

The Bacterial Transcription Termination Factor Rho Coordinates Mg2+ Homeostasis with Translational Signals

Kriner

Groisman

2015

Journal of Molecular Biology

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The bacterial protein Rho triggers transcription termination at the ends of many operons and when transcription and translation become uncoupled. In addition to these genome-wide activities, Rho implements regulation of specific genes by dictating whether RNA polymerase terminates transcription within the 5’ leader region or continues into the downstream coding region. Here, we report that the Mg2+ channel gene corA in Salmonella enterica serovar Typhimurium, which was previously thought to be constitutively expressed, is regulated by a Rho-dependent terminator located within its 5’ leader region. We demonstrate that the unusually long and highly conserved corA leader mRNA can adopt two mutually exclusive conformations that determine whether or not Rho interacts with a Rho utilization (rut) site on the nascent RNA and thereby prevents transcription of the corA coding region. The RNA conformation that promotes Rho-dependent termination is favored by efficient translation of corL, a short open reading frame located within the corA leader. Thus, corA transcription is inversely coupled to corL translation. This mechanism resembles those governing expression of Salmonella’s other two Mg2+ transport genes, suggesting that Rho links Mg2+ uptake to translational signals.

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“…Increased Mg 2+ uptake may aid in restoring optimal protein synthesis because Mg 2+ acts as a structural component of ribosomes, promotes assembly of initiation complexes, and (as Mg 2+ -ATP) supplies energy for charging of tRNAs [81]. In accordance with this notion, ribosomes and ATP together chelate the majority of Mg 2+ in the cell [81], indicating that sufficient intracellular Mg 2+ pools are essential for continuous translation. Indeed, Mg 2+ starvation of E. coli leads to translational arrest and, ultimately, to depletion of the ribosome pool [82].…”

Section: Rho As a Sensor Of Translational Statusmentioning

confidence: 99%

Learning from the Leaders: Gene Regulation by the Transcription Termination Factor Rho

Kriner

Sevostyanova

Groisman

2016

Trends in Biochemical Sciences

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The RNA helicase Rho triggers 20–30% of transcription termination events in bacteria. While Rho is associated with most transcription elongation complexes, it only promotes termination of a subset. Recent studies of individual Rho-dependent terminators located within the 5′ leader regions of bacterial mRNAs have identified novel mechanisms that govern Rho target specificity and revealed unanticipated physiological functions for Rho. In particular, the multistep nature of Rho-dependent termination enables regulatory input from determinants beyond the sequence of the Rho loading site and allows a given Rho-dependent terminator to respond to multiple signals. Further, the unique position of Rho as a sensor of cellular translation has been exploited to regulate transcription of genes required for protein synthesis, including those specifying Mg2+ transporters.

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When Too Much ATP Is Bad for Protein Synthesis

Cited by 98 publications

References 110 publications

Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media

Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media

The Bacterial Transcription Termination Factor Rho Coordinates Mg2+ Homeostasis with Translational Signals

Learning from the Leaders: Gene Regulation by the Transcription Termination Factor Rho

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