1. Reducing Ribosome Biosynthesis Promotes Translation during Low Mg2+ Stress 
Mauricio H. Pontes, Jinki Yeom, Eduardo A. Groisman 
Molecular Cell 
Volume 64, Issue 3, Pages (November 2016)
DOI: /j.molcel
Copyright © Terms and Conditions

2. Molecular Cell 2016 64, 480-492DOI: (10.1016/j.molcel.2016.05.008)
Copyright © Terms and Conditions

3. Figure 1 Cellular Response to Cytosolic Mg2+ Starvation
(A) Schematic depicting the regulatory circuit controlling ribosome production when Mg2+ is abundant. Mg2+ mediates the assembly of ribosomal components into functional ribosomes. Translation consumes energy and amino acids. A decrease in energy or amino acids decreases expression of ribosomal components.
(B) Cartoon representation showing the role of Mg2+ and ATP in the synthesis of rRNA, assembly of ribosomes, and translation when free cytosolic Mg2+ is abundant.
(C) Schematic depicting the effect of disturbances on the regulatory circuit depicted in (A) due to insufficient free cytosolic Mg2+. This condition impairs ribosome assembly and, therefore, translation, creating an abundance of energy and amino acids, which promotes expression of ribosomal components.
(D) Cartoon representation highlighting the effects of insufficient free cytosolic Mg2+ on ribosome synthesis, assembly, and translation, resulting in the accumulation of non-functional ribosomes (in gray).
(E) Schematic depicting a regulatory circuit that enables cells to repress the synthesis of ribosomal components when free cytosolic Mg2+ is limited. This circuit integrates information about the availability of Mg2+ ions for ribosomal stabilization into the regulatory circuit governing ribosome biosynthesis.
(F) Cartoon representation illustrating the proteins comprising the S. enterica serovar Typhimurium circuit depicted in (E). Mg2+ uptake into the cytosol by the Mg2+ transporters MgtA and MgtB, and a decrease in ATP promoted by the MgtC protein, enables formation of functional ribosomes that are lower in number than when bacteria have abundant Mg2+ (A and B).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © Terms and Conditions

4. Figure 2
The PhoP-Activated Genes mgtA, mgtB, and mgtC Repress rrs Transcription during Cytosolic Mg2+ Starvation
(A) Primer extension analyses of wild-type Salmonella (14028s) grown in high (10 mM) Mg2+, and wild-type (14028s) and phoP (MS7953) Salmonella grown in low (10 μM) Mg2+.
(B) Primer extension analyses of wild-type (14028s), phoP (MS7953), mgtA (EG16735), mgtB (EL5), mgtC (EL4), mgtA mgtB (EG16741), mgtA mgtC (EG16740), and mgtB mgtC (EL6) Salmonella following growth in low Mg2+.
(C) Cytosolic Mg2+ concentration in wild-type (14028s), mgtA mgtB (EG17048), and mgtA mgtB (EG17048) Salmonella harboring the mgtA-expressing plasmid (pMgtA), the mgtB-expressing plasmid (pMgtB), or the plasmid vector (pUHE-21-2-lacIq) during growth in low Mg2+. Cytosolic Mg2+ concentrations were obtained using a genetic reporter (Figures S4A and S4B; Cromie et al., 2006). Values represent the normalized activities of this Mg2+ genetic reporter (i.e., β-galactosidase activity values from Figure S4C were normalized as 1/[pPlac1-6-mgtAleader-lacZ/ pPlac1-6r-lacZ]) and are depicted in arbitrary units (a.u.).
(D) ATP levels of wild-type (14028s), mgtA (EG16735), mgtB (EL5), mgtC (EL4), mgtA mgtB (EG16741), and mgtA mgtB (EG16741) Salmonella harboring pMgtA, pMgtB, or the plasmid vector (pUHE-21-2-lacIq).
(E) Primer extension analyses of wild-type (14028s), mgtA mgtB (EG17048), and mgtA mgtB (EG17048) Salmonella harboring the ATPase expressing plasmid (pATPase) or the plasmid vector (pCP44). Bacteria were grown in N-minimal medium containing high (10 mM) or low (10 μM) Mg2+. Measurements were taken following 3 (high Mg2+) and 6 hr (low Mg2+) growth (OD600 ≈ 0.4–0.6). Error bars represent SDs. Graphs are the average of three to four independent experiments. Primer extension analyses are representative of two to four independent experiments.
(F) RNA/protein ratio of wild-type (14028s) and phoP (MS7953) Salmonella grown in low Mg2+. See also Figures S1, S2, and S4.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © Terms and Conditions

5. Figure 3
MgtC Represses rrs Transcription by Reducing ATP Levels and Promoting ppGpp Accumulation during Cytosolic Mg2+ Starvation
(A) Primer extension analyses of wild-type (14028s), mgtC (EL4), and mgtC (EL4) Salmonella harboring the ATPase expressing plasmid pATPase or the plasmid vector (pCP44) during growth in low (10 μM) Mg2+. For rrs primer extension, lanes 1/2 and 3/4 correspond to lanes 1/2 and 7/8 of the gel image displayed in Figure S7A, respectively.
(B) Quantification of ppGpp levels in wild-type (14028s), mgtC (EL4), and mgtC (EL4) strains of Salmonella harboring the mgtC-expressing plasmid (pMgtC) or the plasmid vector (pUHE-21-2-lacIq) during growth in low Mg2+. Error bars represent SDs. Graph shows the average of four independent experiments.
(C) Primer extension analyses of wild-type (14028s), mgtC (EL4), relA spoT (MP342), and mgtC relA spoT (MP343) Salmonella growth in MOPS glucose medium containing low (10 μM) Mg2+. For rrs primer extension, lanes 1/2 and 3/4 correspond to lanes 1/2 and 5/6 of the gel image displayed in Figure S7B, respectively.
(D) Primer extension analyses of wild-type (14028s), mgtC (EL4), and mgtC relA (MP335) Salmonella grown in low Mg2+, in the presence/absence of serine hydroxamate (Shx, 50 μg/mL).
(E) Primer extension analyses of wild-type (14028s), mgtC (EL4), mgtC rpoBL533P (MP571), and mgtC rpoBΔ532A (MP570) Salmonella during growth in low Mg2+.
(F) Primer extension analyses of wild-type (14028s), mgtC (EL4), and mgtC (EL4) Salmonella with pATPase or the plasmid vector (pCP44) during growth in low (10 μM) Mg2+, in the presence/absence of 50 μg/mL Shx. Unless stated otherwise, strains were grown in N-minimal medium. All measurements were taken following 6 hr of growth in low Mg2+ (OD600 ≈ 0.4–0.6). Primer extension analyses are representative of three to four independent experiments. See also Figures S1–S3 and S7.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © Terms and Conditions

6. Figure 4
MgtA, MgtB, and MgtC Promote Ribosome Assembly, thereby Enabling Translation during Cytosolic Mg2+ Starvation
(A) Polysome analyses of wild-type (14028s), mgtC (EL4), and mgtC (EL4) Salmonella harboring the mgtC-expressing plasmid (pMgtC), or the plasmid vector (pUHE-21-2-lacIq), mgtA mgtB (EG16741), and mgtA mgtB (EG16741) Salmonella harboring the mgtA-expressing plasmid (pMgtA) or the plasmid vector (pUHE-21-2-lacIq) during growth in low (10 μM) Mg2+. Cells were collected for polysome analysis following 6 hr (low Mg2+) of growth (OD600 ≈ 0.4–0.6). Polysome profiles are representative of four independent experiments.
(B) Quantification of L-azidohomoalanine (AHA) labeling of wild-type (14028s), mgtC (EL4), and mgtC (EL4) Salmonella harboring pMgtC or the plasmid vector (pUHE-21-2-lacIq) following growth in low (10 μM) Mg2+.
(C) Quantification of AHA labeling of wild-type (14028s), mgtA mgtB (EG16741), and mgtA mgtB (EG16741) Salmonella harboring pMgtA or the plasmid vector (pUHE-21-2-lacIq) during growth in low Mg2+. Measurements were taken following 6 hr growth in low Mg2+ N-minimal medium lacking methionine (OD600 ≈ 0.4–0.6). Error bars represent SDs. Graphs are the average of four independent experiments. See also Figures S5 and S6.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © Terms and Conditions

7. Figure 5
MgtA, MgtB, and MgtC Readjust Cellular Translation Rates during Cytosolic Mg2+ Starvation
(A) Growth curve of wild-type Salmonella (14028s) in low (10 μM) Mg2+. The arrow indicates the time when MgtA, MgtB, and MgtC are expressed. The color intensity of the legend depicts the concentration of Mg2+ in the cytosol.
(B) Quantification of L-azidohomoalanine (AHA) labeling of wild-type Salmonella (14028s) prior to (2.5 hr) and after (5.5 hr) cytosolic Mg2+ starvation. Error bars represent SDs. Graphs show the average of four independent experiments.
(C) SDS-PAGE analysis of AHA labeling of wild-type (14028s) prior to (2.5 hr) and after (5.5 hr) cytosolic Mg2+ starvation. The gel is representative of four independent experiments. All experiments were carried out in low Mg2+ (10 μM) Mg2+ N-minimal medium lacking methionine.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © Terms and Conditions

8. Figure 6
PhoP/PhoQ Regulates Ribosome Synthesis and Translation in E. coli during Cytosolic Mg2+ Starvation
(A) Time-course cytosolic Mg2+ concentrations of wild-type E. coli grown in medium containing 0 or 10 mM Mg2+. Cytosolic Mg2+ concentrations reflect the normalized β-galactosidase activity values obtained with a genetic reporter for cytosolic Mg2+ and are expressed in arbitrary units (a.u.) (see Experimental Procedures).
(B) Time-course quantification of ATP levels in wild-type (MG1655), phoP (EG12976), mgtA (EG16771), yhiD (MP751), and mgtA yhiD (MP752) E. coli grown in medium lacking Mg2+.
(C) qPCR quantification of rrs transcriptional activity in wild-type (MG1655), phoP (EG12976), mgtA (EG16771), yhiD (MP751), and mgtA yhiD (MP752) E. coli 2 hr following removal of Mg2+ from the growth medium.
(D) Quantification of AHA labeling of wild-type (MG1655), phoP (EG12976), mgtA (EG16771), yhiD (MP751), and mgtA yhiD (MP752) strains of E. coli 2 hr following removal of Mg2+ from the growth medium. All experiments were carried out in MOPS glucose medium. Error bars represent SDs. Graphs show the average of four independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © Terms and Conditions

9. Figure 7
Cellular Response to Cytosolic Mg2+ Starvation in S. enterica serovar Typhimurium
(A) Low extracytoplasmic Mg2+ activates the sensor PhoQ, which promotes the phosphorylation state of the regulator PhoP (PhoP-P). PhoP-P binds to the promoters of mgtA and mgtCB and recruits RNA polymerase. Transcription at these promoters is initiated but does not proceed into the coding regions because cytosolic Mg2+ levels are still high. There is a high abundance of functional ribosomes and translation is efficient.
(B) As cells grow and consume the existing Mg2+ in the environment, levels of free cytosolic Mg2+ eventually drop below a certain threshold. This decrease in free cytosolic Mg2+ impairs ribosomal subunit association, causing a global slow down on translation that promotes transcription elongation into the coding regions of the mgtA, mgtC, and mgtB genes. The Mg2+ transporters MgtA and MgtB import Mg2+ into the cytoplasm, and MgtC decreases ATP levels and promotes ppGpp accumulation. This response increases the levels of free cytosolic Mg2+, but decreases the number of functional of ribosomes, which enables cells to continue translation, albeit at a reduced rate.
(C) Mutants unable to elicit the response presented in (B) (i.e., phoP, phoQ, mgtC, and mgtA mgtB) are unable to maintain the pool of free cytosolic Mg2+ and are thus defective to assemble their ribosomes and have much reduced translation.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © Terms and Conditions