Volume 27, Issue 12, Pages 1757-1767.e5 (June 2017) Fatty Acid Availability Sets Cell Envelope Capacity and Dictates Microbial Cell Size  Stephen Vadia, Jessica L. Tse, Rafael Lucena, Zhizhou Yang, Douglas R. Kellogg, Jue D. Wang, Petra Anne Levin  Current Biology  Volume 27, Issue 12, Pages 1757-1767.e5 (June 2017) DOI: 10.1016/j.cub.2017.05.076 Copyright © 2017 Elsevier Ltd Terms and Conditions

Current Biology 2017 27, 1757-1767.e5DOI: (10.1016/j.cub.2017.05.076) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 RNA, Protein and Fatty Acid Synthesis Differentially Impact Cell Size (A) Mean square area, length, and width versus mass doublings per hour in six different carbon sources or in LB-glc with subinhibitory concentrations of rifampicin (3, 4, 5, and 6 μg/ml), chloramphenicol (0.5, 1.0, and 1.5 μg/ml), or cerulenin (50, 60, 65, and 70 μg/ml). (B) Percent of cells with FtsZ rings versus doubling times for the above conditions. (C) Mean square area versus mass doublings/hour for E. coli ΔfabH. n ≥ 3 experiments; n ≥ 100 cells measured per experiment. Error bars indicate the SEM. Black lines indicate the line of best fit of data from cells cultured in six different carbon sources. Carbon sources from nutrient rich to nutrient poor include the following: LB-glc, LB, and AB minimal medium ± 0.5% casamino acids with 0.2% glucose or 0.4% succinate. Blue, green, red, and purple lines indicate lines of best fit. Wedges indicate increasing carbon availability or antibiotic concentration. See also Figure S1 and Tables S1 and S2. Current Biology 2017 27, 1757-1767.e5DOI: (10.1016/j.cub.2017.05.076) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 The Addition of Exogenous Fatty Acids Increases Cell Size by Increasing Plasma Membrane Capacity (A) E. coli cell size distributions and mean square areas in LB-glc after inhibition of fatty acid synthesis with 70 μg/ml cerulenin ± 10 μg/ml oleic acid (OA). (B) Mean square areas of wild-type or mutants lacking FadL, FadD, or FadE. OM, outer membrane; IM, inner membrane. (C) E. coli cell size distributions and mean square areas after inhibition of translation with 1.5 μg/ml chloramphenicol in the presence or absence of 10 μg/ml OA. n ≥ 3 experiments; n ≥ 100 cells measured per experiment. All error bars indicate the SEM. See also Figure S2, Tables S4–S6. Current Biology 2017 27, 1757-1767.e5DOI: (10.1016/j.cub.2017.05.076) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Fatty Acid Synthesis Is a Conserved Determinant of Cell Size (A) Mean length versus mass doublings per hour for B. subtilis in LB-glc with subinhibitory concentrations of rifampicin (1.25, 2.5, 5, and 7.5 ng/ml), chloramphenicol (0.6, 0.7, and 0.8 μg/ml), or cerulenin (2, 3, 3.5, and 4 μg/ml). (B) B. subtilis cell size distributions and mean square areas in LB-glc after inhibition of fatty acid synthesis with 3.5 μg/ml cerulenin ± 2 μg/ml OA. (C) S. cerevisiae cell size distributions and mean square areas in YEPD medium after inhibition of fatty acid synthesis with 0.22 μg/ml cerulenin or cerulenin + 50 μg/ml OA. n ≥ 3 experiments; n ≥ 100 cells measured per experiment. All error bars indicate the SEM. See also Figure S2 and Tables S3 and S4. Current Biology 2017 27, 1757-1767.e5DOI: (10.1016/j.cub.2017.05.076) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Cell Size Increases with Phospholipid-Available Fatty Acid Content (A) Overproduction of FadR increases E. coli cell size. (B) Representative phase contrast images of E. coli wild-type or pFadR cells. Scale bar, 5 μm. (C) Cell size distribution versus fadR induction. (D) Cell size versus growth rate after induction of fadR. The black line indicates line of best fit for E. coli cultured in different carbon sources (reproduced from Figure 1A). The red line indicates line of best fit for pFadR cells. (E) Relative amounts of phospholipid-available fatty acids per cell, as determined by GC-FID (micrograms of fatty acids) and Petroff-Hausser counts (cells/milliliter). (F) Electron micrographs of wild-type and pFadR cells. Scale bar, 100 nm. n ≥ 3 experiments; n ≥ 100 cells measured per experiment for cell size measurements. Error bars indicate the SEM. The color key indicates different IPTG concentrations and applies to (A), (C), (D), and (E). Asterisks indicate significant differences between conditions. See also Figures S3–S5 and Table S7. Current Biology 2017 27, 1757-1767.e5DOI: (10.1016/j.cub.2017.05.076) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Expression of the Cytosolic Thioesterase ’tesA Diverts Fatty Acids from Phospholipid Synthesis and Negates FadR-Mediated Increases in Cell Size (A) Schematic showing the products formed after reaction of ’TesA and PlsB with long-chain acyl-ACPs. PlsB transfers acyl chains from long-chain acyl-ACPs to the 1 position of glycerol-3-phosphate to form lysophosphatidic acid (LPA). ’TesA is a leaderless variant of TesA that localizes to the cytoplasm, where it competes for long-chain acyl-ACPs available for fatty acid synthesis, cleaving them to generate free fatty acids that are released into the culture medium. (B) Mean square area of E. coli after induction of pFadR, p’TesA, or pFadR/p’TesA. n ≥ 3 experiments; n ≥ 100 cells measured per experiment. All error bars indicate the SEM. See also Table S7. Current Biology 2017 27, 1757-1767.e5DOI: (10.1016/j.cub.2017.05.076) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 Fatty Acid Synthesis and Nutrient Availability Impact Size Independent of ppGpp (A) Histogram of E. coli overproducing the ppGpp synthase RelA alone (pRelA) and in combination with the transcriptional activator FadR (pRelA/pFadR). (B) Cell size versus growth rate of wild-type MG1655 and an isogenic ppGpp0 strain in LB-glc, LB, AB-caa-glc, and AB-caa-succ for both strains, as well as AB-glc and AB-succ for the wild-type strain. (The ppGpp0 strain cannot grow without the addition of casamino acids). Black and gray lines indicate lines of best fit for wild-type and ppGpp0 datasets, respectively. n ≥ 3 experiments; n≥ 100 cells measured per experiment. Error bars indicate the SEM. See also Figures S6 and S4 and Tables S1 and S7. Current Biology 2017 27, 1757-1767.e5DOI: (10.1016/j.cub.2017.05.076) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 7 ppGpp Is Required to Preserve Membrane Integrity and Cell Viability in the Absence of Fatty Acid Synthesis (A) Colony-forming units versus time for wild-type MG1655 and ppGpp0 strains. Cells were grown to OD600 of ∼0.4 in LB-glc, treated with 500 μg/ml of cerulenin (at t = 0), and plated on LB agar every 30 min for 240 min. (B) Mean fluorescence intensity versus time of wild-type and ppGpp0 cells treated with cerulenin in the presence of propidium iodide. (C) Images of cells sampled at selected time points from (B). Intense fluorescence is indicative of loss of membrane integrity. Scale bar, 2 μm. (D) ppGpp serves as a linchpin coupling lipid synthesis and membrane capacity to other aspects of anabolic metabolism. In nutrient-rich medium, ppGpp levels are low, allowing for increased rates of transcription and translation that support rapid expansion of volume and cell envelope capacity. Inhibition of lipid synthesis increases ppGpp levels, downregulating other biosynthetic processes to balance different aspects of anabolic metabolism. In the absence of ppGpp, cells are unable to downregulate other anabolic pathways in response to defects in lipid synthesis, causing cell volume to outpace cell envelope capacity, which leads to cell lysis. See also Figure S7. Current Biology 2017 27, 1757-1767.e5DOI: (10.1016/j.cub.2017.05.076) Copyright © 2017 Elsevier Ltd Terms and Conditions