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DnaC inactivation in Escherichia coli K-12 induces the SOS response and expression of nucleotide biosynthesis genes.

Løbner-Olesen A, Slominska-Wojewodzka M, Hansen FG, Marinus MG - PLoS ONE (2008)

Bottom Line: Transcription of the dnaA and dnaC genes was increased at the non-permissive temperature in the respective mutant strains indicating auto-regulation of both genes.Induction of the SOS regulon was observed in dnaC2 cells at 38 degrees C and 42 degrees C.Flow cytometric analysis revealed that dnaC2 mutant cells at non-permissive temperature had completed the early stages of chromosome replication initiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Science, Systems and Models, Roskilde University, Roskilde, Denmark.

ABSTRACT

Background: Initiation of chromosome replication in E. coli requires the DnaA and DnaC proteins and conditionally-lethal dnaA and dnaC mutants are often used to synchronize cell populations.

Methodology/principal findings: DNA microarrays were used to measure mRNA steady-state levels in initiation-deficient dnaA46 and dnaC2 bacteria at permissive and non-permissive temperatures and their expression profiles were compared to MG1655 wildtype cells. For both mutants there was altered expression of genes involved in nucleotide biosynthesis at the non-permissive temperature. Transcription of the dnaA and dnaC genes was increased at the non-permissive temperature in the respective mutant strains indicating auto-regulation of both genes. Induction of the SOS regulon was observed in dnaC2 cells at 38 degrees C and 42 degrees C. Flow cytometric analysis revealed that dnaC2 mutant cells at non-permissive temperature had completed the early stages of chromosome replication initiation.

Conclusion/significance: We suggest that in dnaC2 cells the SOS response is triggered by persistent open-complex formation at oriC and/or by arrested forks that require DnaC for replication restart.

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Relative gene expression of heat shock genes in the wildtype, dnaA46 and dnaC2 strains.Bacteria were grown at 30°C, 38°C or 42°C for 90 min, harvested and total RNA extracted for microarray analysis. The groS and groL genes, but not rpoH, are part of the heat-shock regulon.
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pone-0002984-g002: Relative gene expression of heat shock genes in the wildtype, dnaA46 and dnaC2 strains.Bacteria were grown at 30°C, 38°C or 42°C for 90 min, harvested and total RNA extracted for microarray analysis. The groS and groL genes, but not rpoH, are part of the heat-shock regulon.

Mentions: The fluorescent labeled RNA or cDNA from the E. coli MG1655 strains was scanned exactly as described in the Affymetrix User Guide (www.affymetrix.com) and analyzed using GeneChip Analysis Suite software. Raw data were exported as text files and imported into Microsoft Excel (Office 2003) in which further sorting was accomplished. In the comparison of 4,242 gene encoding proteins in the dnaA and dnaC strains, we required that all genes should have a “present call”. Furthermore, we required that the actual numbers compared in the analysis should be above a (more or less arbitrarily chosen) threshold. For the comparison analyses shown in the Figures 2, 3, 5, 6 of this article we chose 200 scanning units as the minimal threshold. Using this threshold value, we typically obtained present calls for between 3200 to 4000 genes. The average expression for a given probe set (including those with an absence call or negative value) was 385 scanning units and the highest expressed genes (encoding ribosomal proteins and other components of the protein synthesis apparatus) were around 5000 scanning units. The results are from duplicate trials. All data from the microarray analysis can be found at http://users.umassmed.edu/martin.marinus/arrays/.


DnaC inactivation in Escherichia coli K-12 induces the SOS response and expression of nucleotide biosynthesis genes.

Løbner-Olesen A, Slominska-Wojewodzka M, Hansen FG, Marinus MG - PLoS ONE (2008)

Relative gene expression of heat shock genes in the wildtype, dnaA46 and dnaC2 strains.Bacteria were grown at 30°C, 38°C or 42°C for 90 min, harvested and total RNA extracted for microarray analysis. The groS and groL genes, but not rpoH, are part of the heat-shock regulon.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2500167&req=5

pone-0002984-g002: Relative gene expression of heat shock genes in the wildtype, dnaA46 and dnaC2 strains.Bacteria were grown at 30°C, 38°C or 42°C for 90 min, harvested and total RNA extracted for microarray analysis. The groS and groL genes, but not rpoH, are part of the heat-shock regulon.
Mentions: The fluorescent labeled RNA or cDNA from the E. coli MG1655 strains was scanned exactly as described in the Affymetrix User Guide (www.affymetrix.com) and analyzed using GeneChip Analysis Suite software. Raw data were exported as text files and imported into Microsoft Excel (Office 2003) in which further sorting was accomplished. In the comparison of 4,242 gene encoding proteins in the dnaA and dnaC strains, we required that all genes should have a “present call”. Furthermore, we required that the actual numbers compared in the analysis should be above a (more or less arbitrarily chosen) threshold. For the comparison analyses shown in the Figures 2, 3, 5, 6 of this article we chose 200 scanning units as the minimal threshold. Using this threshold value, we typically obtained present calls for between 3200 to 4000 genes. The average expression for a given probe set (including those with an absence call or negative value) was 385 scanning units and the highest expressed genes (encoding ribosomal proteins and other components of the protein synthesis apparatus) were around 5000 scanning units. The results are from duplicate trials. All data from the microarray analysis can be found at http://users.umassmed.edu/martin.marinus/arrays/.

Bottom Line: Transcription of the dnaA and dnaC genes was increased at the non-permissive temperature in the respective mutant strains indicating auto-regulation of both genes.Induction of the SOS regulon was observed in dnaC2 cells at 38 degrees C and 42 degrees C.Flow cytometric analysis revealed that dnaC2 mutant cells at non-permissive temperature had completed the early stages of chromosome replication initiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Science, Systems and Models, Roskilde University, Roskilde, Denmark.

ABSTRACT

Background: Initiation of chromosome replication in E. coli requires the DnaA and DnaC proteins and conditionally-lethal dnaA and dnaC mutants are often used to synchronize cell populations.

Methodology/principal findings: DNA microarrays were used to measure mRNA steady-state levels in initiation-deficient dnaA46 and dnaC2 bacteria at permissive and non-permissive temperatures and their expression profiles were compared to MG1655 wildtype cells. For both mutants there was altered expression of genes involved in nucleotide biosynthesis at the non-permissive temperature. Transcription of the dnaA and dnaC genes was increased at the non-permissive temperature in the respective mutant strains indicating auto-regulation of both genes. Induction of the SOS regulon was observed in dnaC2 cells at 38 degrees C and 42 degrees C. Flow cytometric analysis revealed that dnaC2 mutant cells at non-permissive temperature had completed the early stages of chromosome replication initiation.

Conclusion/significance: We suggest that in dnaC2 cells the SOS response is triggered by persistent open-complex formation at oriC and/or by arrested forks that require DnaC for replication restart.

Show MeSH
Related in: MedlinePlus