The CcrM DNA methyltransferase of Agrobacterium tumefaciens is essential, and its activity is cell cycle regulated.

DNA methylation is now recognized as a regulator of multiple bacterial cellular processes. CcrM is a DNA adenine methyltransferase found in the alpha subdivision of the proteobacteria. Like the Dam enzyme, which is found primarily in Escherichia coli and other gamma proteobacteria, it does not appear to be part of a DNA restriction-modification system. The CcrM homolog of Agrobacterium tumefaciens was found to be essential for viability. Overexpression of CcrM is associated with significant abnormalities of cell morphology and DNA ploidy. Mapping of the transcriptional start site revealed a conserved binding motif for the global response regulator CtrA at the -35 position; this motif was footprinted by purified Caulobacter crescentus CtrA protein in its phosphorylated state. We have succeeded in isolating synchronized populations of Agrobacterium cells and analyzing their progression through the cell cycle. We demonstrate that DNA replication and cell division can be followed in an orderly manner and that flagellin expression is cyclic, consistent with our observation that motility varies during the cell cycle. Using these synchronized populations, we show that CcrM methylation of the chromosome is restricted to the late S phase of the cell cycle. Thus, within the alpha subdivision, there is a conserved cell cycle dependence and regulatory mechanism controlling ccrM expression.

Evolutionarily conserved promoter motifs and enhancer organization in the mouse gene encoding the IIB myosin heavy chain isoform expressed in adult fast skeletal muscle.

We have isolated the mouse gene for the MHC isoform expressed in adult type IIB (fast-contracting, glycolytic) skeletal muscle fibers, and determined the DNA sequence of the promoter region. This sequence represents the first example of a promoter for a gene encoding an adult-specific isoform of a mammalian skeletal MHC. The proximal 200 bp of the promoter contains several sequence motifs which are identical or very similar to homologous motifs found in the promoters of a family of chicken skeletal MHC genes. Of these, two novel AT-rich sequences may be important for regulation of the promoter. A second feature of the mouse IIB MHC promoter sequence concerns a number of sequence motifs located at ca. -1,000 bp which are organized in a similar fashion in the IIB MHC promoter and a homologous region of promoter of the mouse muscle creatine kinase gene.

Influence of uvrB and pKM101 on the spectrum of spontaneous, UV- and gamma-ray-induced base substitutions that revert hisG46 in Salmonella typhimurium.

Oligonucleotide probes were used to identify base substitutions in 1089 revertants of hisG46 in Salmonella typhimurium that arose spontaneously or following irradiation with UV- or gamma-rays. The hisG46 allele, carrying a mutant CCC codon (Pro) in place of the wild-type codon CTC (Leu69) reverted via 6 distinguishable mutational events--C to T transitions at codon sites 1 or 2, C to A or C to G transversions at codon site 1, C to A at codon site 2, and an extragenic suppressor mutation. The distribution of hisG46 revertants differed among treatments and was influenced by the DNA-repair capacity of the bacteria. Plasmid pKM101 enhanced the frequencies of both spontaneous and induced mutations; transversion events were enhanced more efficiently by pKM101 than were transition events. Compared to Uvr+ bacteria, Uvr- bacteria had higher frequencies of spontaneous and induced mutations; transition mutations were enhanced more efficiently than were transversion mutations. The influence of DNA-repair activities on the mutational spectra provides some insights on the origins of spontaneous and UV-induced mutations.