Oscillations in expression of a negatively autoregulated gene in E. coli are synchronized to the cell cycle by gene doubling Zach Hensel, Tatiana T. Márquez-Lago Integrative Systems Biology Unit
Feedback-dependent oscillations
400
300
400
100 200 300 Elapsed time (min)
400
-2σ
[mVenus]
0
100
100
100
200
200
200
+2σ
PR
OR1
NF
NF
lac
300
300
20 mm Gel pad 1 mm Objective
Imaging
Glass slide Spacer #1 coverslip
515
NF NFΔcro,lac λ attB
-0.02 -0.04 -0.06
Single-cell timelapse microscopy 150’
165’
190’
205’
220’
2 µm
NFΔcro,lac
120’
135’
150’
165’
190’
205’
NF
220’
2 µm
● Colonies grown from single cells for hundreds of minutes. ● Chromosome (nucleoid) localization for NFlac indicates mVenus-Cro non-specific DNA binding. ● Cytoplasmic localization for NFΔcro,lac. ● 15 separate colonies imaged in each experiment at 5-minute intervals; experiments replicated for all strains. ● Two-color imaging facilitates automated cell segmentation.
mVenus-Cro concentration
135’
NFlac
120’
E. coli MG1655
oriC ori
terC NF
ter1
0.2 0.4 0.6 0.8 Fraction of cell cycle elapsed
+2σ 0 -2σ NFori 0 100 200 300 400 500 +2σ 0 -2σ NFter1 0 100 200 300 400 500 +2σ 0 -2σ NFter2 0 100 200 300 400 Elapsed time (min)
0.8
NFΔcro,lac NFlac NFori NFter1 NFter2
0.6 0.4 0.2
1
0
50
Possible biomolecular mechanisms? ● New synthetic biology methods simplify chromosome integration3—potential advantage over plasmid-borne constructs from strong copy-number control. ● Models of chromosome-integrated synthetic constructs must accurately account for gene doubling. ● Synchronization of oscillations to gene doubling time suggests new mechanisms must be taken into account: Concentration Copy number
Repression probability
ter1
Copy number
-100 0 100 Time lag (min)
250
1
10-1
10-4 Frequency (Hz)
10-3
Discussion
NF
-200
200
10
● Power spectral density calculated as the FFT of mVenus concentration autocorrelation. Strains with negative feedback exhibit increased noise bandwidth with power at higher frequencies1,2.
Elapsed time
0.05 0 -0.05
100 150 Time lag (min)
NFΔcro,lac NFlac NFori NFter1 NFter2
● Relatively weak cell-cycle dependence for NFΔcro,lac.
NFlac NFΔcro,lac
0.05 0 -0.05
Mean (µ) and Fano factor (µ2/σ) in molecules/µm3. Coefficient of variation (µ2/σ2) is unitless. N is number of colonies used in data analysis. Statistics compiled from two separate experiments for each strain. Loci listed relative to 4.6-Mbp E. coli MG1655 genome (Accession #NC_000913.3).
● Autocorrelation calculated for all single-cell trajectories and averaged. Slow autocorrelation decay for NFΔcro,lac consistent with individual trajectories and cell lineages. Damped oscillations for some strains with negative feedback.
NFori
0.05 0 -0.05
1
● Cell-cycle-averaged mVenus-Cro concentration for NFlac is oscillatory with magnitude of ~10% the mean concentration.
cell division
0.05 0 -0.05
0.8
cell cycle frequencies
100 200 300 400 Elapsed time (min)
~doubling time
NFter2
-0.4 -0.2 0 0.2 0.4 0.6 Normalized mVenus concentration
1
0
Integration site determines oscillation phase
● Laser microscopy: Detection down to single-molecule level.
-0.6
● Fano factor is greatly reduced by negative feedback; range of Fano factors may indicate different feedback strengths1.
NF NFΔcro,lac
0.02
● Long-lived memory of high/low mVenus concentration in the absence of negative feedback for NFΔcro,lac.
lac
-0.8
● Negative feedback reduces mean mVenus concentration and increases coefficient of variation.
lac
0.04
400 0
-1
400
● Correlated oscillations in related cells in NFlac mVenus-Cro concentration lineages.
● NFΔcro,lac: Only mVenus; no-feedback construct.
CCD
100 200 300 Elapsed time (min)
1
NFter2 200
● Negative-feedback construct integrated at additional sites proximal to chromosome replication origin and terminus. ● No clear oscillations for one terminal integration construct, NFter2. ● Cross correlation between binary, cell-cycle time series (1 at division times; 0 at other times) and mVenus-Cro concentration exhibits phase shifts corresponding to estimated gene doubling times.
Elapsed time Supercoil density Copy number Elapsed time
References 1 2 3 4 5
Bacteriophage λ lysis/lysogeny 0.06 0.04
cI cro 60
0.02 0
40
-0.02 20
-0.04 -0.06
0.2 0.4 0.6 0.8 Fraction of cell cycle elapsed
1
PR
PRM
80 Lysis probability4
445
×1.6
● Well-characterized components from bacteriophage λ: allows rational change of circuit properties. ● NFlac: Functional mVenus-Cro fluorescent protein fusion binds OR1 to repress its own transcription.
561
Leica DMI6000b
0
Cell-cycle cross correlation
Gel pad Cells Spacer
● Chromosome-integrated, synthetic circuits in E. coli.
1.5
0.5
● Long-lived excursions from mean concentration without feedback for NFΔcro,lac.
400
0.06
OR1 RBS ttgactattttacctctggcggtgataatggttgcAAGTACTAAGGAGGTTATTATATG PR –35 PR –10
Sample preparation
400
300
Δcro,lac
● Oscillations observed with negative feedback for NFlac.
400
200 300 Elapsed time (min)
Δcro,lac
mVenus RBS
400
cI
oriC
cro
OR3 OR2 OR1
lytic genes
HK022 21/e14 Atlas/ɸ80
300
100
300
E. coli MG1655
terC
P2 HP1
NFΔcro,lac mVenus concentration
400
200
Statistics for all single-cell time series
λ
200
300
100
2
NFΔcro,lac NFlac NFori NFter1 NFter2
Power spectral density (Hz-1)
100
200
400
2.5
● Single-cell mVenus concentration tracked for each possible trajectory.
P22 NFlac
OR1
cro
100
200
300
Average mVenus concentration
mVenus RBS
NF
100
200
[mVenus-Cro] -2σ +2σ
Methods Strain development NFlac PR
0
100
+2σ 0 -2σ 0 +2σ 0 -2σ 0 +2σ 0 -2σ 0 +2σ 0 -2σ 0 12000 8000 4000
mVenus molecules/cell
mVenus-Cro molecules/cell
NFlac mVenus-Cro concentration
+2σ 0 -2σ 0 +2σ 0 -2σ 0 +2σ 0 -2σ 0 +2σ 0 -2σ 0 1200 800 400
No Feedback
Average mVenus concentration
Negative feedback
Normalized autocorrelation
Synthetic biologists create genetic circuits from well-characterized components such as transcriptional repressors. Oscillatory expression of transcriptional repressors in single-gene negative-feedback circuits has been attributed to high cooperativity, long chemical reaction delays, and fast protein degradation. Using single-cell timelapse experiments in E. coli, we observed oscillatory expression of the bacteriophage λ repressor Cro in a simple circuit lacking these attributes. Strong oscillations required negative feedback and occurred in synchrony with the cell cycle. Integrating the circuit at different genomic loci produced phase shifts that predictably correlated with gene location, suggesting that chromosome replication can drive regulated oscillatory gene expression. Consistent with theoretical models, introducing negative feedback increased the coefficient of variation, reduced the Fano factor, and increased the noise bandwidth of Cro expression. Our results should be accounted for in the design of chromosome-integrated genetic networks. Furthermore, cell-cycle-periodic expression of Cro suggests an evolutionary basis for site-specific λ integration.
Gene expression noise Frequency
Abstract
● Lysis/lysogeny decision-making determined by mutual repression by cI and cro. Lysis probability increases with cell age4—possible selective benefit as older/larger cells produce more bacteriophage particles. ● Site-specific integration loci of lamboid phages biased towards late gene-doubling5. Our results suggest that this will favor Cro expression and possibly lysis in older cells. ● Negative transcriptional regulation by competitive DNA binding is a common regulatory motif—a simple mechanism for synchronizing gene expression to the cell cycle?
Simpson, M. L., Cox, C. D. & Sayler, G. S. Frequency domain analysis of noise in autoregulated gene circuits. PNAS 100 (2003). Austin, D. W. et al. Gene network shaping of inherent noise spectra. Nature 439 (2006). St-Pierre, F. et al. One-step cloning and chromosomal integration of DNA. ACS Synthetic Biology 2 (2013). St-Pierre, F. & Endy, D. Determination of cell fate selection during phage λ infection. PNAS 105 (2008). Campbell, A. M. Chromosomal insertion sites for phages and plasmids. J Bacteriol 174 (1992).
Acknowledgements
We thank Jie Xiao for providing plasmids used in making constructs (pJB106, pZH051)
Contacts:
[email protected] [email protected] 