Survival During Nutrient Starvation

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Figure 1 : Survival and physiological parameters for heterogeneous mutant populations during starvation conditions. The entire collection of haploid deletion mutants was starved for either phosphate (+) or leucine (x) in the presence (solid line) or absence (dashed line) of kanamycin for nearly 500 hours following an initial period of 24 hours of batch growth. (A) Survival of replicate populations grown in media of identical composition except for the limiting nutrient. Survival of each population was monitored by determining viability of the population at each time point by counting CFUs on rich media plates. (B) The total number of cells/mL was determined at each time point and remained essentially unchanged throughout the starvation regime. (C) Culture biomass was estimated using a Klett colorimeter and showed a gradual increase for populations starved for phosphate and gradual decrease when populations were starved for leucine. (D) The average cell volume, measured using a Coulter counter, showed a gradual increase for populations starved for phosphate and a slow decline for populations starved for leucine.
Figure 2 : Development and validation of quantitative barcode sequencing for multiplexed mutant screens. We tested the error and variance associated with each step of our protocol. . (A) By sequencing a single barcode we found that 98% of sequences up to sequencing cycle 23 (gray dotted line) perfectly match the expected sequence (n = 2,340,984). (B) Additional cycles of PCR introduce minimal variation in the estimated proportions of mutants. The best-correlated estimates of mutant abundance are found between 15-25 PCR cycles (increasingly black values approach a correlation of 1.0; the minimum correlation is 0.94). (C) Resequencing the same PCR product from a complex mixture of mutants on two different flow cells yields highly reproducible results (Pearson correlation = 0.99; n = 3,329). (D) Complete technical replicates of quantitative barcode sequencing (i.e. independent DNA preparations, PCR and sequencing reactions) are highly reproducible (Pearson correlation = 0.94; n = 3,439).
Figure 3 : Experimental design for multiplexed mutant survival analysis using quantitative barcode sequencing. We constructed normalized pools of the yeast haploid deletion collection by growing individual mutants on rich media (YDP) plates and pooling mutants in liquid YPD for archival purposes. A 1.6mL aliquot of the unselected pooled mutants was used to inoculate (t=0) cultures limited for either phosphate or leucine. The starvation period commenced after 24 hours of culture growth. At each time point we removed a 1mL sample from the culture and expanded the viable sub-population by allowing 24 hours of outgrowth in supplemented minimal media. DNA was isolated from the resulting culture and analyzed using quantitative barcode sequencing.
Figure 4 : Population diversity decline and mutant abundance profiles during prolonged starvation. (A) We determined the number of unique strains identified through barcode sequencing at each time point for populations starved for phosphate (gray bars) or leucine (black bars). (B) Hierarchical clustering of mutant abundance profiles during starvation experiments. We clustered vectors of relative abundance in the population normalized by the abundance of each mutant at t = 24 hours (log2 transformed). Black indicates that the strain has not changed in abundance. Yellow represents increases in abundance and blue represents decreases in abundance. Failure to detect the strain in the population is indicated by gray. We identified clusters of mutants that were either specifically (C) decreased in relative abundance upon phosphate starvation or (D) increased in relative abundance upon leucine starvation. Several mutants are decreased in relative abundance under both starvation conditions including (E) a cluster including several autophagy gene mutants.
Figure 5 : Quantitative analysis of absolute death rates during prolonged starvation. We calculated the absolute rate of death for mutants using measurements of population viability and estimates of relative strain abundance using quantitative barcode sequencing of uptags (circles) and downtags (triangles) for the putatively neutral HO0 allele starved for phosphate (A) and leucine (B). The data presented are barcode counts normalized between all uptag or downtag sequencing results. A value of 1 was added to all normalized barcode counts prior to log2 transformation. The rate of death was determined using a generalized linear model for uptag (long dash line) and downtag (short dash line) data. These rates were compared to rates calculated from independent data (viable cells / L)obtained for the isogenic strain BY4742 (crosses) subjected to starvation in pure cultures. We calculated death rates for all mutants in each starvation condition and converted these values to half-lives for all barcode data that yielded a significant death rate (FDR <5%). The distribution of half-lives for mutants starved for phosphate is centered around 289 hours (C) and 22.8 hours (D) for leucine starved mutants. The half-life of the HO0 strain when starved for phosphate (blue dotted line) or leucine (red dotted line in both histograms) is shown for reference.
Figure 6 : Functional gene modules altering survival during nutrient starvation. The distributions of half-lives for subsets of genes defined by different methods of categorization were compared with the overall distribution of half-lives (white bars) in each experiment shown. (A) Phosphate starvation, mitochondrion organization (blue, Go Slim term, n = 281, p = 5.88 x 10-41)), peroxisome organization (red, GO:0007031, n = 27, p = 0.00013) (B) Leucine starvation, mitochondrion organization (blue, Go Slim term, n = 276, p = 9.23 x 10-14), peroxisome organization (red, GO:0007031, n = 25, p = 4.63 x10-5) (C) Phosphate starvation, autophagy (blue, GO:0006914, n = 51, p = 2.0 x 10-13), translation (red, GO:0006412, n = 305, p = 1.46 x 10-12) (D) Leucine starvation, autophagy (blue, GO:0006914, n = 50, p = 0.00014), translation (red, GO:0006412, n = 301, p = 2.33 x 10-11) (E) Phosphate starvation, mRNA processing (blue, GO:0006397, n = 91, p = 3.38 x 10-11), mRNA transport (red, GO:0051028, n = 42, p = 6.45 x 10-5) (F) Leucine starvation, mRNA processing (blue, GO:0006397, n = 91, p = 0.87), mRNA transport (red, GO:0051028, n = 40, p = 0.87) (G) Phosphate starvation, chromatin modification (blue, GO:0016568, n = 89, p = 2.86 x 10-6), histone acetyltransferase complex (red, SGD defined protein complex, n = 41, p = 8.82 x 10-5) (H) Leucine starvation, chromatin modification (blue, GO:0016568, n = 90, p = 0.032), histone acetyltransferase complex (red, SGD defined protein complex, n = 40, p = 0.16) (I) Phosphate starvation, cytoskeleton (blue, GO:0005856, n = 151, p = 2.03 x 10-5), microtubule organizing center (red, SGD defined protein complex, n = 30, p = 3.16 x 10-5) (J) Leucine starvation, cytoskeleton (blue, GO:0005856, n = 148, p = 0.14), microtubule organizing center (red, SGD defined protein complex, n = 30, p = 0.75) (K) Phosphate starvation, slow growth in YPD (blue, defined by (GIAEVER et al. 2002), n = 637, p =2.46 x 10-103), impaired growth in rapamycin (red, defined by (DUDLEY et al. 2005), n = 137, p = 1.16 x 10-19) (L) Leucine starvation, slow growth in YPD (blue, defined by (GIAEVER et al. 2002), n = 619, p = 3.04 x 10-16), impaired growth in rapamycin (red, defined by (DUDLEY et al. 2005), n = 132, p = 0.00058) (M) Phosphate starvation, MRPL10 cluster of the yeast metabolic cycle (blue, defined in (TU et al. 2005), n = 54, p = 1.62 x 10-16) (N) Leucine starvation, MRPL10 cluster of the yeast metabolic cycle (blue, defined in (TU et al. 2005), n = 53, p = 4.8 x 10-6).

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