Distribution of fluorescence values of cells w/ LacY::YFP Cells begin to show “all or nothing” behavior at about uM TMG. Fluorescence intensity is on the X axis, number of cells is on the Y axis

Images of “all or nothing” behavior at 30 um TMG in LacY::YFP cells Note that the “nothing” cells have a few LacY::YFP molecules

Most “nothing” cells have 0-4 LacY::YFP molecules

~400 molecules of LacY::Yfp are needed for induction in 40 uM TMG Measurement of the threshold of permease molecules for induction. (A) Single-cell time traces of fluorescence intensity, normalized by cell size, starting from different initial permease numbers. The initial LacY-YFP numbers were prepared through dilution by cell division of fully induced cells after removal of the inducer. Upon adding 40mM TMG at time 0, those cells with low initial permease numbers lost fluorescence with time as a result of dilution by cell division and photo bleaching,whereas those cells with high initial permease numbers exhibited an increase in fluorescence as a result of reinduction. Permease molecule numbers were estimated from cell fluorescence(28). The dashed red line indicates the determined threshold. (B) The probability of induction of a cell within 3 hours as a function of the initial permease number was determined using traces from 90 cells.The probability of induction (p) was fit with a Hill equation p = y4.5/(y ) for the initial permease number y. The threshold of permease numbers for induction was thus determined tobe375molecules. Error bars are the inverse square root oft he sample size at each point.

Novick has something to contribute to the analysis of data in Choi et al.

Cells are rapidly induced in high level of TMG The slope is a measurement of speed of induction. It is proportional to the amount of b-gal activity made per generation.

Induction in low levels of TMG is slower Slope =.005

After one generation cells should be ~30% induced and induction should be quick Should begin to see an increase in expression right away at 40 uM TMG (at least 30% induced in one generation). Go back to slide 1 and see that MUCH less than 30% of the cells were induced after 24 hours in the 40 uM TMG experiment.

If lac can’t loop, then induction occurs more readily C: With O3 removed the lac region can’t loop. Such cells are able to induce lacY even when they start out with very low numbers of LacY initially. Compare to Fig 2A where ~400 LacY molecules were needed for induction to occur. D: DNA looping is needed for bistability. Without looping population are not bimodal—compare with first slide.

LacY is made in bursts-small and large The Experiment: done in lacY- cells with 200 uM TMG; the lac promoter is driving another membrane protein (Tsr) fused to YFP. The Results: Cells rarely make new LacYs and when they do, they only make a few (top panel). Every once and a while they make many, over an extended period (bottom panel).

When lac region can’t loop large bursts are common In panel D, note that the x-axis has MUCH higher values—cells w/o lac looping make have many bursts that make a lot of product relative to cells that still loop the lac region (panel C)

Model for expression bursts At high TMG concentrations, TMG binds to LacI and causes it to fall off of the DNA. Once off of the DNA, it tends to stay off, leading to large bursts of transcription. Note that the Km for TMG binding to DNA-bound LacI is ~1mM (that is when internal TMG concentrations are ~1mM, LacI has a 50:50 chance of binding TMG and falling off of the DNA

Model for expression bursts (cont) At low TMG concentrations one side of the LacI tetramer will fall off of O1 and a small burst of transcription will take place before rebinding. If the whole tetramer should fall off (this is rare), then it will bind to TMG and stay off, giving a large burst. Note that the Km for TMG binding to DNA-bound LacI is ~1mM BUT its Km for LacI not bound to DNA is ~ 10 uM. This means that 40 uM TMG is not enough to bump LacI off of the operators, but it is enough to keep it off once it falls off on its own. In strains w/o lac looping, every time LacI falls off one operator it finds itself in solution and quickly bound by TMG. This tends to make every burst a large burst.

Experimental set up used in Robert et al. This allows them to follow induction in single cells by microscopy as they grow.

Typical (?) results Figure 2 Establishment of bimodality in microcolonies. (A) Fluorescent images (YFP channel) of a microcolony of LCY1 just before TMG introduction (inset) and 2 h. (B) Fluorescence intensity of all individual cells (black symbols) and mean fluorescence of the population (yellow) as a function of time. The green and red lines are representative examples of single-cell trajectories. The dashed gray line indicates the time of TMG introduction. (C) Distribution of YFP fluorescence of single cells 2 h after TMG introduction

Induction levels of single cells and their descendents were tracked over time. Cells appeared to be correlated— descendents of one cell often behaved the same way all inducing or not inducing. The circled cell shows one cell whose daughters all induced, even though the two daughters were separated from each other before TMG was added (at the dashed line. Figure 3 Typical ‘Lineage history tree’ established for the microcolony presented in Figure 2A. Individual cells are plotted as horizontal lines where the color corresponds to fluorescence intensity (YFP channel) as a function of time (horizontal axis). At division time, a vertical line is drawn to connect the mother cell and its two daughters. The dashed line corresponds to TMG introduction. The black circle indicates an example of division of a cluster’s common ancestor before TMG introduction.

Parents tend to leave descendants that are all either “On” or “Off” For each cell present at time tI (TMG introduction), the proportion of induced cells in its final progeny was calculated. The distribution of these proportions exhibits two peaks at 0 and 1 (Figure 3B, left), suggesting that most of the initial cells have in their progeny either almost only induced cells or almost only uninduced cells.

Statistical evidence that induction is correlated in descedents Figure 4 (A) Typical genealogical tree with pairs of sister cells present at TMG introduction (red) and pairs of sister cells in the population of mothers (blue) and grandmothers (green) of cells present at TMG introduction. The vertical black line corresponds to TMG introduction. (B) Correlation of responses between sister cells and first and second cousin cells (from four different experiments). The mean fluorescence of the progeny of a cell is plotted against the mean fluorescence of its sister’s progeny, for cells present at TMG introduction (red) or cells in the population of mothers (blue) and grandmothers (green, inset) of cells present at TMG introduction

Show movie!!

What caused correlated behavior? Why might all the descendents of the circled cell be readily induced? The authors show 2 things influence the correlated behavior: 1. LacI levels in the initial (circled) cell. Less LacI in a cell means that its descendants are more likely to induce in response to low levels of TMG. This could be caused by larger/longer bursts in cells with little LacI. 2. Growth rate: slow growth of an initial cell also makes it more likely that descendants will induce.

Paper by Elowitz et al.

Effects of bursting and stochastic expression Bursting mRNA levels Protein levels Effect on other genes

Signal noise and gene regulation noise affect gene expression

Paper by Elowitz et al. Extrinsic noise: N ext. Noise in a population of cells that that arises from global conditions that can vary from cell to cell. Such global conditions may be levels of RNA polymerase, internal concentration of inducer, internal concentration of important regulatory proteins. Intrinsic noise: N int. This is noise that would come about even if there was no extrinsic noise. Elowitz et al. point out that N int can be seen as the variation in expression of 2 identical promoters in the same cell. A Nint value of.25 means that expression of 2 such promoters would vary by 25%. N tot =(Standard deviation of expression)/(mean of the expression) N tot 2 =N ext 2 + N int 2

High expression levels lead to low noise CFP (colored green) and YFP (colored red) each driven by plac(O1) in a lacI- cell Ntot=.077 Next=.054 Nint=.055 CFP (colored green) and YFP (colored red) each driven by plac(O1) in a lacI+ cell with IPTG Ntot=.117 Next=.098 Nint=.063

Low expression levels lead to high noise (~25x dimmer than cells w/ IPTG) CFP (colored green) and YFP (colored red) each driven by plac(O1) in a lacI+ cell with no IPTG Ntot=.41 Next=.33 Nint=.25 CFP (colored green) and YFP (colored red) each driven by plac(O1) in a lacI+ cell with no IPTG Ntot=.37 Next=.32 Nint=.19

Noise in two stains, one noisy, the other less so M22: recA+, D22 recA+

Noise as a function of expression levels Increasing fluorescence/expression 

Cell death by antibiotics- a typical experiment

-if you regrow the persisters, they are normally resistant, and give rise to a small percentage of persisters---so their resistance is not due to mutation.

Persisters -slow growing or non-growing cells that are resistant to: antibiotics (causes problems clinically) heat, dessication phage other stresses -usually make up a small percentage of a population -the actually percentage is a function of how frequently the environment changes -mutants can be found that make persisters more often. One classic persister++ mutation is in the gene hipA

--> if HipA is made by itself, cells cannot grow!

The hipA gene was put under control of the tetracycline repressor -when tetracycline was added to cells, they made HipA in proportion to the amount of tet added.

The amount of HipA effects when colonies appear (ie affects when cells start to grow) -Show movie growth rate is the same for all levels of HipA High HipA causes colonies to appear late and with large variability in appearance time

+Amp  HipA madeNo HipA made see persisters only when HipA is made. Only slow appearing cells are resistant!

The effects of high HipA can be seen on plates too-it inhibits the start of growth -cell with highest amount of HipA does not divide until late

HipA inhibition happens at a threshold (blue arrow w/o HipB, orange arrow w/ HipB)

Cells above HipA threshold put off growth, cells below grow right away

Model red = free HipA

How might this happen? How might there be enough free HipA to trigger various levels of persistance?

Low TMG data from Novick Note that at 10 uM TMG 43% of the cells are induced after several generations. Choi et al. had very few induced cells after 24 hours at 30 uM!!