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Gene location and DNA density determine transcription factor distributions in Escherichia coli.

Kuhlman TE, Cox EC - Mol. Syst. Biol. (2012)

Bottom Line: Contrary to expectation, we find that the distribution depends on the spatial location of its encoding gene.We demonstrate that the spatial distribution of LacI is also determined by the local state of DNA compaction, and that E. coli can dynamically redistribute proteins by modifying the state of its nucleoid.We propose a model for intranucleoid diffusion that can reconcile these results with previous measurements of LacI diffusion, and we discuss the implications of these findings for gene regulation in bacteria and eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ, USA. tkuhlman@illinois.edu

ABSTRACT
The diffusion coefficient of the transcription factor LacI within living Escherichia coli has been measured directly by in vivo tracking to be D = 0.4 μm(2)/s. At this rate, simple models of diffusion lead to the expectation that LacI and other proteins will rapidly homogenize throughout the cell. Here, we test this expectation of spatial homogeneity by single-molecule visualization of LacI molecules non-specifically bound to DNA in fixed cells. Contrary to expectation, we find that the distribution depends on the spatial location of its encoding gene. We demonstrate that the spatial distribution of LacI is also determined by the local state of DNA compaction, and that E. coli can dynamically redistribute proteins by modifying the state of its nucleoid. Finally, we show that LacI inhomogeneity increases the strength with which targets located proximally to the LacI gene are regulated. We propose a model for intranucleoid diffusion that can reconcile these results with previous measurements of LacI diffusion, and we discuss the implications of these findings for gene regulation in bacteria and eukaryotes.

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Average TF distributions as a function of growth state: two chromatids. Average DNA (first row) and LacI-Venus distributions with and without the DNA binding domain (subsequent groups of two rows, respectively) are shown as a function of lacI-venus gene integration location (rows) for exponentially slow growing cells in M63+0.5% glycerol (left) and exponentially fast growing cells containing two chromatids (right). Fast growth in RDM+0.5% glucose yields a doubling time of 22±2 min. Stationary phase results are from cells grown overnight in M63 minimal medium+0.5% glycerol. LacI-Venus distributions of exponentially slow growing origin and terminus integrants we obtained by Venus excitation with a 488-nm wavelength laser; all other conditions were obtained with a 514-nm laser to increase the signal-to-noise ratio. Average DNA content in each case is scaled relative to slow growing cells containing one chromatid in Figure 4. Distributions of gene location shown in the first column are from growth in M63+0.5% glycerol that are 4.2–4.5 μm in length; gene distributions in exponential fast growth are similar and are compared directly in Supplementary Figure 5B.
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f4: Average TF distributions as a function of growth state: two chromatids. Average DNA (first row) and LacI-Venus distributions with and without the DNA binding domain (subsequent groups of two rows, respectively) are shown as a function of lacI-venus gene integration location (rows) for exponentially slow growing cells in M63+0.5% glycerol (left) and exponentially fast growing cells containing two chromatids (right). Fast growth in RDM+0.5% glucose yields a doubling time of 22±2 min. Stationary phase results are from cells grown overnight in M63 minimal medium+0.5% glycerol. LacI-Venus distributions of exponentially slow growing origin and terminus integrants we obtained by Venus excitation with a 488-nm wavelength laser; all other conditions were obtained with a 514-nm laser to increase the signal-to-noise ratio. Average DNA content in each case is scaled relative to slow growing cells containing one chromatid in Figure 4. Distributions of gene location shown in the first column are from growth in M63+0.5% glycerol that are 4.2–4.5 μm in length; gene distributions in exponential fast growth are similar and are compared directly in Supplementary Figure 5B.

Mentions: E. coli changes its morphology and the gross compaction state of its nucleoid in response to changing growth conditions. We assayed the spatial distribution of LacI-Venus in a variety of growth states to determine whether these changes in the intracellular environment affect the steady-state distribution of LacI-Venus protein. The results for cells containing an equal total amount of DNA in different growth states are shown in Figures 3 and 4. When bulk growth is arrested in stationary phase cells generally contain a single chromosome, are morphologically small, and the DNA is densely packed (Figure 3, second column; Supplementary Figure 3). As the growth rate increases, cells become larger and the DNA becomes more loosely packed (Figure 3, third column; Supplementary Figure 3). As cells reach their maximum growth rate of ∼20 min/doubling they contain at least two or more complete sister chromatids (Figure 4, third column; Supplementary Figure 3), and the trend of DNA decondensation in fast growing cells continues compared with slower growing cells late in the life cycle which also contain two complete sister chromatids (Figure 4, second column).


Gene location and DNA density determine transcription factor distributions in Escherichia coli.

Kuhlman TE, Cox EC - Mol. Syst. Biol. (2012)

Average TF distributions as a function of growth state: two chromatids. Average DNA (first row) and LacI-Venus distributions with and without the DNA binding domain (subsequent groups of two rows, respectively) are shown as a function of lacI-venus gene integration location (rows) for exponentially slow growing cells in M63+0.5% glycerol (left) and exponentially fast growing cells containing two chromatids (right). Fast growth in RDM+0.5% glucose yields a doubling time of 22±2 min. Stationary phase results are from cells grown overnight in M63 minimal medium+0.5% glycerol. LacI-Venus distributions of exponentially slow growing origin and terminus integrants we obtained by Venus excitation with a 488-nm wavelength laser; all other conditions were obtained with a 514-nm laser to increase the signal-to-noise ratio. Average DNA content in each case is scaled relative to slow growing cells containing one chromatid in Figure 4. Distributions of gene location shown in the first column are from growth in M63+0.5% glycerol that are 4.2–4.5 μm in length; gene distributions in exponential fast growth are similar and are compared directly in Supplementary Figure 5B.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472691&req=5

f4: Average TF distributions as a function of growth state: two chromatids. Average DNA (first row) and LacI-Venus distributions with and without the DNA binding domain (subsequent groups of two rows, respectively) are shown as a function of lacI-venus gene integration location (rows) for exponentially slow growing cells in M63+0.5% glycerol (left) and exponentially fast growing cells containing two chromatids (right). Fast growth in RDM+0.5% glucose yields a doubling time of 22±2 min. Stationary phase results are from cells grown overnight in M63 minimal medium+0.5% glycerol. LacI-Venus distributions of exponentially slow growing origin and terminus integrants we obtained by Venus excitation with a 488-nm wavelength laser; all other conditions were obtained with a 514-nm laser to increase the signal-to-noise ratio. Average DNA content in each case is scaled relative to slow growing cells containing one chromatid in Figure 4. Distributions of gene location shown in the first column are from growth in M63+0.5% glycerol that are 4.2–4.5 μm in length; gene distributions in exponential fast growth are similar and are compared directly in Supplementary Figure 5B.
Mentions: E. coli changes its morphology and the gross compaction state of its nucleoid in response to changing growth conditions. We assayed the spatial distribution of LacI-Venus in a variety of growth states to determine whether these changes in the intracellular environment affect the steady-state distribution of LacI-Venus protein. The results for cells containing an equal total amount of DNA in different growth states are shown in Figures 3 and 4. When bulk growth is arrested in stationary phase cells generally contain a single chromosome, are morphologically small, and the DNA is densely packed (Figure 3, second column; Supplementary Figure 3). As the growth rate increases, cells become larger and the DNA becomes more loosely packed (Figure 3, third column; Supplementary Figure 3). As cells reach their maximum growth rate of ∼20 min/doubling they contain at least two or more complete sister chromatids (Figure 4, third column; Supplementary Figure 3), and the trend of DNA decondensation in fast growing cells continues compared with slower growing cells late in the life cycle which also contain two complete sister chromatids (Figure 4, second column).

Bottom Line: Contrary to expectation, we find that the distribution depends on the spatial location of its encoding gene.We demonstrate that the spatial distribution of LacI is also determined by the local state of DNA compaction, and that E. coli can dynamically redistribute proteins by modifying the state of its nucleoid.We propose a model for intranucleoid diffusion that can reconcile these results with previous measurements of LacI diffusion, and we discuss the implications of these findings for gene regulation in bacteria and eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ, USA. tkuhlman@illinois.edu

ABSTRACT
The diffusion coefficient of the transcription factor LacI within living Escherichia coli has been measured directly by in vivo tracking to be D = 0.4 μm(2)/s. At this rate, simple models of diffusion lead to the expectation that LacI and other proteins will rapidly homogenize throughout the cell. Here, we test this expectation of spatial homogeneity by single-molecule visualization of LacI molecules non-specifically bound to DNA in fixed cells. Contrary to expectation, we find that the distribution depends on the spatial location of its encoding gene. We demonstrate that the spatial distribution of LacI is also determined by the local state of DNA compaction, and that E. coli can dynamically redistribute proteins by modifying the state of its nucleoid. Finally, we show that LacI inhomogeneity increases the strength with which targets located proximally to the LacI gene are regulated. We propose a model for intranucleoid diffusion that can reconcile these results with previous measurements of LacI diffusion, and we discuss the implications of these findings for gene regulation in bacteria and eukaryotes.

Show MeSH
Related in: MedlinePlus