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Adaptation in bacterial flagellar and motility systems: from regulon members to 'foraging'-like behavior in E. coli.

Zhao K, Liu M, Burgess RR - Nucleic Acids Res. (2007)

Bottom Line: Bacterial flagellar motility and chemotaxis help cells to reach the most favorable environments and to successfully compete with other micro-organisms in response to external stimuli.To define the physiological role of these two regulators, we carried out transcription profiling experiments to identify, on a genome-wide basis, genes under the control of these two regulators.In addition, the synchronized pattern of increasing CRP activity causing increasing FlhDC expression with decreasing carbon source quality, together with the apparent coupling of motility activity with the activation of motility and chemotaxis genes in poor quality carbon sources, highlights the importance of CRP activation in allowing E. coli to devote progressively more of its limited reserves to search out better conditions.

View Article: PubMed Central - PubMed

Affiliation: McArdle Laboratory for Cancer Research, Department of Genetics, University of Wisconsin, Madison, WI 53706, USA.

ABSTRACT
Bacterial flagellar motility and chemotaxis help cells to reach the most favorable environments and to successfully compete with other micro-organisms in response to external stimuli. Escherichia coli is a motile gram-negative bacterium, and the flagellar regulon in E. coli is controlled by a master regulator FlhDC as well as a second regulator, flagellum-specific sigma factor, sigma(F). To define the physiological role of these two regulators, we carried out transcription profiling experiments to identify, on a genome-wide basis, genes under the control of these two regulators. In addition, the synchronized pattern of increasing CRP activity causing increasing FlhDC expression with decreasing carbon source quality, together with the apparent coupling of motility activity with the activation of motility and chemotaxis genes in poor quality carbon sources, highlights the importance of CRP activation in allowing E. coli to devote progressively more of its limited reserves to search out better conditions. In adaptation to a variety of carbon sources, the motile bacteria carry out tactical responses by increasing flagellar operation but restricting costly flagellar synthesis, indicating its capability of strategically using the precious energy in nutrient-poor environments for maximizing survival.

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Carbon source effects on cell motility through activation of CRP. (A) SDS–PAGE gel shows purified CRP protein as well as MultiMark Standard and native gel shift assays show the binding of CRP to upstream DNA fragment of FlhDC operon. The palindromic consensus DNA-binding site for CRP dimer is shown in red. (B) The transcript abundance of two CRP-dependent genes, cstA and cpdB, in cells grown on different carbon sources. (C) The transcript abundance of flhD, fliA and crp in cells grown on different carbon sources. (D) The transcript abundance of σF-dependent genes, motA, tar and fliC, in cells grown on different carbon sources. Note, these carbon sources are of differing quality as defined by the resulting log-phase growth rates which are 0.97 generation h−1 in glucose, 0.50 generation h−1 in succinate, 0.34 generation h−1 in alanine, 0.21 generation h−1 in acetate and 0.13 generation h−1 in proline.
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Figure 5: Carbon source effects on cell motility through activation of CRP. (A) SDS–PAGE gel shows purified CRP protein as well as MultiMark Standard and native gel shift assays show the binding of CRP to upstream DNA fragment of FlhDC operon. The palindromic consensus DNA-binding site for CRP dimer is shown in red. (B) The transcript abundance of two CRP-dependent genes, cstA and cpdB, in cells grown on different carbon sources. (C) The transcript abundance of flhD, fliA and crp in cells grown on different carbon sources. (D) The transcript abundance of σF-dependent genes, motA, tar and fliC, in cells grown on different carbon sources. Note, these carbon sources are of differing quality as defined by the resulting log-phase growth rates which are 0.97 generation h−1 in glucose, 0.50 generation h−1 in succinate, 0.34 generation h−1 in alanine, 0.21 generation h−1 in acetate and 0.13 generation h−1 in proline.

Mentions: Relief from the carbon catabolite repression is a complex regulatory circuit that triggers reprogramming of global gene expression patterns to adapt the changes in external environment. This mechanism will activate the cyclic AMP receptor protein (CRP) (43), a global transcriptional factor that positively regulates most carbon catabolic pathways. While it is known that carbon catabolite repression affects the flagellar synthesis and the CRP activation might be involved in alleviating this repression (41,42,44,45), much less is known regarding the role of CRP in motility regulation under a range of carbon source conditions. To determine the effect of different carbon sources on E. coli CRP activity as well as the functional relevance between the active CRP level and the expression of FlhDC operon in a range of conditions with the sequenced E. coli strain MG1655, we performed the following assays. CRP protein was purified using the pET expression system (Supplemental Material, Figure S2) for in vitro assays. In electrophoretic mobility-shift assays as shown in Figure 5A, the upstream DNA fragment of flhDC operon can be shifted by purified CRP protein. CRP is a dimer of identical subunits. The consensus tandem DNA-binding site for CRP dimer has been identified by in silico analysis as shown in red color which is approximately palindromic and provides two very similar recognition sites, one for each subunit of the dimer. For sequenced E. coli strain MG1655, there are 250 bp between the CRP-binding sites and FlhDC translational start site, which has a rare translation start codon with GTG that is different from another E. coli strain studied by Soutourina et al. (44). We then measured the transcriptional levels of two well-known CRP positively controlled genes (cstA and cpdB) (to represent the level of active CRP in the cell) as well as the expression level of flhDC operon by the quantitative RT-PCR approaches (Figure 5B and C). Compared with cells grown in glucose, the transcriptional levels of CRP-dependent genes gradually increased in succinate, alanine and acetate grown cells, with a slight decline in proline compared with the level in acetate. The same change in the transcriptional pattern of the FlhDC operon in cells grown in alternative carbon sources relative to that in glucose-grown cells can be observed as shown in Figure 5C. In addition, we made a cyaA in-frame deletion strain. cyaA encodes adenylate cyclase that is required to catalyze the formation of cyclic AMP (46). The pattern of increasing the transcriptional levels of CRP-dependent genes in low-quality carbon sources, as was observed in the wild-type strain, disappeared in the cyaA deficient strain (as shown in Supplemental Material, Figure S3). This indicates that CRP-cAMP plays an important role in promoter activation in our assays. No significant change of the transcriptional level of CRP was observed (Figure 5C), suggesting that the induction of CRP-dependent genes might be mainly due to the activation of CRP rather than the increase in CRP expression (47–49).Figure 5.


Adaptation in bacterial flagellar and motility systems: from regulon members to 'foraging'-like behavior in E. coli.

Zhao K, Liu M, Burgess RR - Nucleic Acids Res. (2007)

Carbon source effects on cell motility through activation of CRP. (A) SDS–PAGE gel shows purified CRP protein as well as MultiMark Standard and native gel shift assays show the binding of CRP to upstream DNA fragment of FlhDC operon. The palindromic consensus DNA-binding site for CRP dimer is shown in red. (B) The transcript abundance of two CRP-dependent genes, cstA and cpdB, in cells grown on different carbon sources. (C) The transcript abundance of flhD, fliA and crp in cells grown on different carbon sources. (D) The transcript abundance of σF-dependent genes, motA, tar and fliC, in cells grown on different carbon sources. Note, these carbon sources are of differing quality as defined by the resulting log-phase growth rates which are 0.97 generation h−1 in glucose, 0.50 generation h−1 in succinate, 0.34 generation h−1 in alanine, 0.21 generation h−1 in acetate and 0.13 generation h−1 in proline.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Carbon source effects on cell motility through activation of CRP. (A) SDS–PAGE gel shows purified CRP protein as well as MultiMark Standard and native gel shift assays show the binding of CRP to upstream DNA fragment of FlhDC operon. The palindromic consensus DNA-binding site for CRP dimer is shown in red. (B) The transcript abundance of two CRP-dependent genes, cstA and cpdB, in cells grown on different carbon sources. (C) The transcript abundance of flhD, fliA and crp in cells grown on different carbon sources. (D) The transcript abundance of σF-dependent genes, motA, tar and fliC, in cells grown on different carbon sources. Note, these carbon sources are of differing quality as defined by the resulting log-phase growth rates which are 0.97 generation h−1 in glucose, 0.50 generation h−1 in succinate, 0.34 generation h−1 in alanine, 0.21 generation h−1 in acetate and 0.13 generation h−1 in proline.
Mentions: Relief from the carbon catabolite repression is a complex regulatory circuit that triggers reprogramming of global gene expression patterns to adapt the changes in external environment. This mechanism will activate the cyclic AMP receptor protein (CRP) (43), a global transcriptional factor that positively regulates most carbon catabolic pathways. While it is known that carbon catabolite repression affects the flagellar synthesis and the CRP activation might be involved in alleviating this repression (41,42,44,45), much less is known regarding the role of CRP in motility regulation under a range of carbon source conditions. To determine the effect of different carbon sources on E. coli CRP activity as well as the functional relevance between the active CRP level and the expression of FlhDC operon in a range of conditions with the sequenced E. coli strain MG1655, we performed the following assays. CRP protein was purified using the pET expression system (Supplemental Material, Figure S2) for in vitro assays. In electrophoretic mobility-shift assays as shown in Figure 5A, the upstream DNA fragment of flhDC operon can be shifted by purified CRP protein. CRP is a dimer of identical subunits. The consensus tandem DNA-binding site for CRP dimer has been identified by in silico analysis as shown in red color which is approximately palindromic and provides two very similar recognition sites, one for each subunit of the dimer. For sequenced E. coli strain MG1655, there are 250 bp between the CRP-binding sites and FlhDC translational start site, which has a rare translation start codon with GTG that is different from another E. coli strain studied by Soutourina et al. (44). We then measured the transcriptional levels of two well-known CRP positively controlled genes (cstA and cpdB) (to represent the level of active CRP in the cell) as well as the expression level of flhDC operon by the quantitative RT-PCR approaches (Figure 5B and C). Compared with cells grown in glucose, the transcriptional levels of CRP-dependent genes gradually increased in succinate, alanine and acetate grown cells, with a slight decline in proline compared with the level in acetate. The same change in the transcriptional pattern of the FlhDC operon in cells grown in alternative carbon sources relative to that in glucose-grown cells can be observed as shown in Figure 5C. In addition, we made a cyaA in-frame deletion strain. cyaA encodes adenylate cyclase that is required to catalyze the formation of cyclic AMP (46). The pattern of increasing the transcriptional levels of CRP-dependent genes in low-quality carbon sources, as was observed in the wild-type strain, disappeared in the cyaA deficient strain (as shown in Supplemental Material, Figure S3). This indicates that CRP-cAMP plays an important role in promoter activation in our assays. No significant change of the transcriptional level of CRP was observed (Figure 5C), suggesting that the induction of CRP-dependent genes might be mainly due to the activation of CRP rather than the increase in CRP expression (47–49).Figure 5.

Bottom Line: Bacterial flagellar motility and chemotaxis help cells to reach the most favorable environments and to successfully compete with other micro-organisms in response to external stimuli.To define the physiological role of these two regulators, we carried out transcription profiling experiments to identify, on a genome-wide basis, genes under the control of these two regulators.In addition, the synchronized pattern of increasing CRP activity causing increasing FlhDC expression with decreasing carbon source quality, together with the apparent coupling of motility activity with the activation of motility and chemotaxis genes in poor quality carbon sources, highlights the importance of CRP activation in allowing E. coli to devote progressively more of its limited reserves to search out better conditions.

View Article: PubMed Central - PubMed

Affiliation: McArdle Laboratory for Cancer Research, Department of Genetics, University of Wisconsin, Madison, WI 53706, USA.

ABSTRACT
Bacterial flagellar motility and chemotaxis help cells to reach the most favorable environments and to successfully compete with other micro-organisms in response to external stimuli. Escherichia coli is a motile gram-negative bacterium, and the flagellar regulon in E. coli is controlled by a master regulator FlhDC as well as a second regulator, flagellum-specific sigma factor, sigma(F). To define the physiological role of these two regulators, we carried out transcription profiling experiments to identify, on a genome-wide basis, genes under the control of these two regulators. In addition, the synchronized pattern of increasing CRP activity causing increasing FlhDC expression with decreasing carbon source quality, together with the apparent coupling of motility activity with the activation of motility and chemotaxis genes in poor quality carbon sources, highlights the importance of CRP activation in allowing E. coli to devote progressively more of its limited reserves to search out better conditions. In adaptation to a variety of carbon sources, the motile bacteria carry out tactical responses by increasing flagellar operation but restricting costly flagellar synthesis, indicating its capability of strategically using the precious energy in nutrient-poor environments for maximizing survival.

Show MeSH
Related in: MedlinePlus