SM: Data curation, Investigation, Methodology, Resources
CL: Formal analysis, Validation
LB: Conceptualization, Data curation, Funding acquisition, Methodology, Resources, Supervision, Writing - original draft, Writing - review & editing
In rich medium, W303
The diauxic shift (DS), which we operationally define as the time point at which all the glucose has been exhausted from the medium, is shown as yellow-filled time points. (A and B) Log and linear plots of optical density of W303 and BY4741 as a function of time. (C) Percent of cells in G1 for the same cultures. (D) Optical density (black squares) and the percent of cells in G1 (blue squares) of W303 measured at 15 minute intervals to better define when cells begin to accumulate in G1 with respect to the DS (yellow filled symbols). (E) BY4741 does not undergo an asymmetrical cell division, but (F) lab strain S288c and wild strain RM11 carry out this asymmetrical division just like W303. (G) The
W303 and BY4741 are two of the most commonly used lab strains of
W303 prototrophs grown in rich medium begin to slow growth and accumulate in G1 before they exhaust all the glucose from the medium and undergo the diauxic shift (DS). They undergo one more asymmetrical division and stop dividing with 95% of the cells in G1 about 24 hours later. These cells reach an optical density (OD
600
) of over 20 at saturation (Figure 1A and B). However, BY4741 achieves an OD
600
and cell number of about one-quarter that attained by W303 prototrophs after 48 hours of growth. The log plot in Figure 1A shows that BY4741 displays an initial logarithmic phase of growth, but little growth takes place after 16 hours (Figure 1B). The DS (yellow symbol) for BY4741 occurs at 20 hours, which is four hours after it ceases mass accumulation, and six hours later than that of W303. The initial increase in 1N DNA from 25% to 50% takes place before the DS, but there is almost no further increase in optical density or G1 DNA content thereafter (Figure 1C). In contrast, W303 undergoes the initial increase to 50% 1N DNA one hour before the DS (Figure 1D), and then reaches 95% about 24 hours later (Figure 1C.) The cell number (p=10
-5
) and percent of cells in G1 (p=10
-4
) is significantly lower in BY4741 than in W303 after 48 hours. In addition, the asymmetric cell division that takes place in W303 after the DS and gives rise to small daughter cells with a modal cell volume of less than 20 femtoliters (Li
To see how the differences between BY4741 and W303 would affect the phenotype of mutants with known defects in G1 arrest, we analyzed
We have observed large differences in the transition to quiescence in two “wild type” lab strains grown in rich medium. However, there are no publicly available, fully sequenced genomes for either of these two related strains to facilitate further understanding of these differences. BY4741 is clearly not limited for glucose when it stops dividing in rich medium and it doesn’t undergo the asymmetric cell division or the G1 arrest typical of other strains. Despite its undeniable importance in studies of log phase growth, BY4741 is not ideal for studies of the transition to quiescence. The W303 prototroph behaves more like wild strains, but it has a truncated
BY6500 is the prototrophic version of W303 (Li
Flow cytometry was processed as stated in (Li
BY6500 |
|
BY7288 |
|
BY4196 |
|
DMA530 |
|
BY6941 |
|
BY6942 |
|
FUNDING Support came from NIGMS R01GM120318 to L.L.B. C.L. was supported by the Howard Hughes Medical Institute as a fellow of the Helen Hay Whitney Foundation in the Biggins lab.