DnaC Inactivation in Escherichia coli K-12 Induces the SOS Response and Expression of Nucleotide Biosynthesis Genes

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 38uC and 42uC. 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

Phenotypic reversal in dam mutants of Escherichia coli K-12 by a recombinant plasmid containing the dam+ gene.

A recombinant plasmid, pMQ3, carrying the dam gene of Escherichia coli K-12, was constructed and transformed into dam+ and dam- strains. Both dam- and dam+ strains containing pMQ3 showed a wild phenotype for all traits, including mutation rate, except for a 10-fold increase in DNA adenine methylase activity

Induction of damage inducible (SOS) repair in dam mutants of Escherichia coli exposed to 2-aminopurine

2-Aminopurine induces damage inducible (SOS) repair in an Escherichia coli dam-4 strain but not in a dam-4 mutS456 derivative or in dam+ bacteria

Correlation of DNA adenine methylase activity with spontaneous mutability in Escherichia coli K-12

Using a multicopy plasmid in which the tac promoter has been placed in front of the dam gene of Escherichia coli K-12, we show that levels of DNA adenine methylase activity are correlated with the spontaneous mutation frequency

Insertion mutations in the dam gene of Escherichia coli K-12

The dam gene of E. coli can be inactivated by insertion of Tn9 or Mud phage. Strains bearing these mutations are viable indicating that the dam gene product is dispensable

An epidemiological study of enteric viruses in sewage with molecular characterization by RT-PCR and sequence analysis.

International audienceThe aim of this study was to assess the presence and seasonal frequency of various enteric viruses in wastewater treatment. The detection of astrovirus, norovirus, enterovirus, hepatitis A virus (HAV) and rotavirus was carried out by molecular analyses in concentrated water samples collected over 18 months at the entrance and exit of an activated sludge sewage treatment plant. The reverse transcriptase-polymerase chain reaction (RT-PCR) results were confirmed by sequencing, and comparative phylogenetic analysis was performed on the isolated strains. Genomes of human astrovirus and human rotavirus were identified in 26/29 and 11/29 samples of raw sewage, respectively, and in 12/29 and 13/29 treated effluent samples, respectively. Some rotavirus sequences detected in environmental samples were very close to those of clinical strains. Noroviruses, enteroviruses and HAV were not detected during the study period. This could be related to the small sample volume, to the sensitivity of the detection methods or to local epidemiological situations. Frequent detection of viral RNA, whether infectious or not, in the exit effluent of sewage treatment indicates wide dispersion of enteric viruses in the environment. Consequently, viral contamination resulting from the use of these treated waters is a risk that needs to be addressed

Kinetic analysis of yersinia pestis DNA adenine methyltransferase activity using a hemimethylated molecular break light oligonucleotide

Background: DNA adenine methylation plays an important role in several critical bacterial processes including mismatchrepair, the timing of DNA replication and the transcriptional control of gene expression. The dependence of bacterial virulenceon DNA adenine methyltransferase (Dam) has led to the proposal that selective Dam inhibitors might function as broadspectrum antibiotics. Methodology/Principal Findings: herein we report the expression and purification of Yersinia pestisDam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that issuitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Daminhibitors. The assay utilised a hemimethylated break light oligonucleotide substrate containing a GATC methylation site.When this substrate was fully methylated by Dam, it became a substrate for the restriction enzyme DpnI, resulting inseparation of fluorophore (fluorescein) and quencher (dabcyl) and therefore an increase in fluorescence. The assays weremonitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisationof Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated forhigh throughput screening, giving a Z-factor of 0.7160.07 indicating that it is a sensitive assay for the identification ofinhibitors. Conclusions/Significance: the assay is therefore suitable for high throughput screening for inhibitors of DNAadenine methyltransferases and the kinetic characterisation of the inhibitio