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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: one chromatid. 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 stationary phase cells (left) and slow growing exponential phase cells containing one chromatid (right). Stationary phase results are from cells grown overnight in M63 minimal medium+0.5% glycerol. LacI-Venus distributions were obtained by excitation with a 488-nm wavelength laser except in the case of the midreplichore integrants, where a 514-nm laser was used for excitation to increase the signal-to-noise ratio. The DAPI channel is shown as green in the first row to improve sensitivity to the eye. Average DNA content for exponential growth is scaled to be directly comparable to Figures 4 and 5 (see Supplementary Figure 3C and D and Kubitschek, 1974). Distributions of gene location in the first column are from cells grown in M63+0.5% glycerol that are 1.9–2.2 μm in length; gene distributions in stationary phase remain similar and are compared directly in Supplementary Figure 5A.
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f3: Average TF distributions as a function of growth state: one chromatid. 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 stationary phase cells (left) and slow growing exponential phase cells containing one chromatid (right). Stationary phase results are from cells grown overnight in M63 minimal medium+0.5% glycerol. LacI-Venus distributions were obtained by excitation with a 488-nm wavelength laser except in the case of the midreplichore integrants, where a 514-nm laser was used for excitation to increase the signal-to-noise ratio. The DAPI channel is shown as green in the first row to improve sensitivity to the eye. Average DNA content for exponential growth is scaled to be directly comparable to Figures 4 and 5 (see Supplementary Figure 3C and D and Kubitschek, 1974). Distributions of gene location in the first column are from cells grown in M63+0.5% glycerol that are 1.9–2.2 μm in length; gene distributions in stationary phase remain similar and are compared directly in Supplementary Figure 5A.

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: one chromatid. 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 stationary phase cells (left) and slow growing exponential phase cells containing one chromatid (right). Stationary phase results are from cells grown overnight in M63 minimal medium+0.5% glycerol. LacI-Venus distributions were obtained by excitation with a 488-nm wavelength laser except in the case of the midreplichore integrants, where a 514-nm laser was used for excitation to increase the signal-to-noise ratio. The DAPI channel is shown as green in the first row to improve sensitivity to the eye. Average DNA content for exponential growth is scaled to be directly comparable to Figures 4 and 5 (see Supplementary Figure 3C and D and Kubitschek, 1974). Distributions of gene location in the first column are from cells grown in M63+0.5% glycerol that are 1.9–2.2 μm in length; gene distributions in stationary phase remain similar and are compared directly in Supplementary Figure 5A.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Average TF distributions as a function of growth state: one chromatid. 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 stationary phase cells (left) and slow growing exponential phase cells containing one chromatid (right). Stationary phase results are from cells grown overnight in M63 minimal medium+0.5% glycerol. LacI-Venus distributions were obtained by excitation with a 488-nm wavelength laser except in the case of the midreplichore integrants, where a 514-nm laser was used for excitation to increase the signal-to-noise ratio. The DAPI channel is shown as green in the first row to improve sensitivity to the eye. Average DNA content for exponential growth is scaled to be directly comparable to Figures 4 and 5 (see Supplementary Figure 3C and D and Kubitschek, 1974). Distributions of gene location in the first column are from cells grown in M63+0.5% glycerol that are 1.9–2.2 μm in length; gene distributions in stationary phase remain similar and are compared directly in Supplementary Figure 5A.
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