Crosslinking of Dam methyltransferase with S-adenosyl-methionine

AbstractHighly purified DNA-adenine methyltransferase was irradiated in the presence of different concentrations of radiolabelled S-adenosyl-methionine (AdoMet) with a conventional Mineralight UV-lamp from several minutes up to 1 h while incubating in ice. Incorporation of radioactivity was monitored by electrophoresis of the crosslink between S-adenosyl-methionine and Dam methylase on SDS-polyacrylamide gels followed by fluorography. Crosslinking reached a maximum in presence of 10 μM S-adenosyl-methionine; it was inhibited in the presence of substates which competitively inhibit methylation of DNA by Dam methylase, like sinefungin or S-adenosyl-homocysteine, but not in the presence of non-inhibitors like ATP or S-isobutyl-adenosine. The crosslink obtained was resistant against a wide range of even drastic conditions commonly used in protein and peptide chemistry. Proteins which do not bind S-adenosyl-methionine, as well as heat activated Dam methylase were not photolabelled. After limited proteolysis the radioactive label appeared only in certain of the peptides obtained. From Western blots carried out with polyclonal antibodies produced against a synthetic peptide corresponding in its sequence to amino acids 92-106 of the Dam methylase, the crosslinking of AdoMet could be tentatively mapped at a position after amino acid 106

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

Expression of the Escherichia coli dam gene

The Escherichia coli dam gene and upstream sequences were cloned from the Kohara phage 4D4. Five promoters were found to contribute to dam gene transcription. P1 and P2 (the major promoter) were situated approximately 3.5 kb upstream of the structural gene, P3 was within the aroB gene, P4 was within the urf74.3 gene, and P5 was in the urf74.3-dam intergenic region. The nucleotide sequence of 2280 bp of DNA containing P1 and P2 was determined and shown to have the potential to encode a protein of approximately 16 kDa between P1, P2 and the aroB gene. This 16 kDa open reading frame has been identified as aroK, the gene for shikimic acid kinase I. Thus the dam gene is part of an operon containing aroK, aroB, urf74.3, and dam. The transcriptional start points of the promoters were determined. A comparison of their nucleotide sequences suggested that P1-P4 were all recognized by the sigma 70 subunit of the RNA polymerase

Novel growth rate control of dam gene expression in Escherichia coli

Transcription of the dam gene in Escherichia coli is growth rate regulated by a mechanism distinct from that used for ribosomal RNA gene promoters. Single-copy operon fusions to lacZ indicated that the major promoter, P2, is responsible for most or all of the growth rate dependence. Promoter P2 is a typical sigma 70 promoter with 18 bp spacing between the -10 and -35 hexamers. Primer extension analysis was used to show that there was no inhibition of transcription from promoter P2 in cells induced for the stringent response. Beta-galactosidase specific activity from a single-copy dam::lacZ fusion was unaffected by either excess rrnB RNA or the level of Fis protein. Thus growth rate control of dam gene expression differs from that of the rRNA and tRNA genes by its lack of response to stringent control, ribosomal feedback and enhanced transcription by Fis protein. We devised a procedure for selection of mutant cells in which dam gene expression was unregulated. One such mutant (cde-4), obtained by miniTn10 insertion, showed the same level of beta-galactosidase activity at all growth rates tested. In contrast, growth rate-dependent expression of the rrnB gene was unaffected by cde-4 confirming the different modes of regulation. The cde-4::miniTn10 insertion is located close to kilobase 670 on the physical map in or near the lipB gene

Growth-rate-dependent transcription initiation from the dam P2 promoter

Transcription of the dam gene in Escherichia coli is dependent on growth rate. Using single-copy promoter::lacZYA fusions we found that of the five promoter regions which affect dam expression, only the P2 promoter shows growth-rate dependence. The determinants for growth-rate control must lie in the region -52 to +27 relative to the transcription start point

Increased adherence and actin pedestal formation by dam-deficient enterohemorrhagic Escherichia coli O157:H7

Enterohaemorrhagic Escherichia coli (EHEC) are highly infectious pathogens capable of causing severe diarrhoeal illnesses. As a critical step during their colonization, EHEC adhere intimately to intestinal epithelial cells and generate F-actin \u27pedestal\u27 structures that elevate them above surrounding cell surfaces. Intimate adhesion and pedestal formation result from delivery of the EHEC type III secretion system (TTSS) effector proteins Tir and EspF(U) into the host cell and expression of the bacterial outer membrane adhesin, intimin. To investigate a role for DNA methylation during the regulation of adhesion and pedestal formation in EHEC, we deleted the dam (DNA adenine methyltransferase) gene from EHEC O157:H7 and demonstrate that this mutation results in increased interactions with cultured host cells. EHECDeltadam exhibits dramatically elevated levels of adherence and pedestal formation when compared with wild-type EHEC, and expresses significantly higher protein levels of intimin, Tir and EspF(U). Analyses of GFP fusions, Northern blotting, reverse transcription polymerase chain reaction, and microarray experiments indicate that the abundance of Tir in the dam mutant is not due to increased transcription levels, raising the possibility that Dam methylation can indirectly control protein expression by a post-transcriptional mechanism. In contrast to other dam-deficient pathogens, EHECDeltadam is capable of robust intestinal colonization of experimentally infected animals