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Escherichia coli str. K-12 substr. W3110

ABSTRACT: Expression of genes at the 45 min time point relative to that at 0 min time after release from initiation-arrest in either presence or absence of TorR

PROVIDER: PRJNA294266 | ENA |

REPOSITORIES: ENA

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Project description:Cell-cycle-dependent temporal transcription of genes is achieved by different mechanisms including a dynamic interaction of activator and repressor proteins with promoters, accumulation (activation) and/or degradation (inactivation) of key regulators as a function of cell cycle. We find that the response regulator TorR protein of the TorR/TorS two-component signal transduction system localizes to the old poles of the Escherichia coli cells, forming a focus. Interestingly, TorR co-localizes with the nucleoid in a cell-cycle-dependent mode, implying a cell-cycle-dependent interaction of TorR with its target genes. The interaction, in turn, regulates transcription of a number of genes associated with Fe2+ transport and Fe-S cluster assembly, also in a cell-cycle-dependent manner. Formation and polar localization of the TorR focus requires the presence of and its interaction with the MreB and DnaK proteins and ATP, suggesting that TorR delivering requires cytoskeleton organization and ATP provided energy. Either the signal receiver or the response regulator domain of TorR protein is also needed for the formation and polar localization of the TorR focus and its interaction with MreB or DnaK. Absence of the protein-protein interactions and ATP leads to a loss in function of TorR as a transcription factor. FtsZ-ring formation seems to be a signal for formation of TorR focus, ensuring each daughter cell has a TorR focus. Therefore, we propose a different mechanism for timing of cell cycle-dependent gene transcription, where a transcription factor interacts with its target genes during a specific period of cell cycle by limiting its own spatial distribution. To address why TorR co-localizes with the nucleoid in a cell-cycle-dependent fashion, we profiled the global transcription patterns using the E. coli Genome GeneChip microarray assay in the presence or absence of TorR protein as a function of the cell cycle. W3110ΔtorRdnaC2 cells carrying pTorR-GFP were synchronized as described above, and shifted down to the permissive temperature and sampled at the 0 min and the 45 min time points for isolation of total RNA during exponential growth. The total RNA was used to synthesize cDNA, and the cDNA was hybridized to Affymetrix GeneChip E. coli Genome 2.0 arrays. The expression level of each gene at the 45 min relative to the 0 min time point was calculated by finding the ratio of values at the 45 min to that at the 0 min. Exponentially growing W3110∆torRdnaC2/pTorR-GFP cells in ABTGcasa medium with or without induction of TorR-GFP expression at the permissive temperature (30oC) were up-shifted to the nonpermissive temperature (42oC) for 2 hours to synchronize the cells. The synchronized cells were down-shifted to the permissive temperature and then cell samples were collected at the 0 and 45 min time points with both the presence and absence of TorR-GFP (presence-Z; absence-W). The total RNA was isolated as mentioned above. The cDNA synthesis, fragmentation, labeling, hybridization of labeled cDNAs from the W3110 derivatives with Affymetrix GeneChip E. coli Genome 2.0 arrays and scanning were performed as described in the Affymetrix User Guide. 6 samples (four replications) and 4 controls (two replications).

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Escherichia coli str. K-12 substr. W3110

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