The CcrM adenine DNA methyltransferase, which specifically modifies GANTC sequences, is necessary for viability in Caulobacter crescentus. To our knowledge, this is the first example of an essential prokaryotic DNA methyltransferase that is not part of a DNA restriction/modification system. Homologs of CcrM are widespread in the Cl subdivision of the Proteobacteria, suggesting that methylation at GANTC sites may have important functions in other members of this diverse group as well. Temporal control of DNA methylation state has an important role in Caulobacter development, and we show that this organism utilizes an unusual mechanism for control of remethylation of newly replicated DNA. CcrM is synthesized de novo late in the cell cycle, coincident with full methylation of the chromosome, and is then subjected to proteolysis prior to cell division.Chromosomal DNA methylation is widespread in prokaryotes and eukaryotes and can affect critical processes such as DNA replication (1, 2), transcription (3-6), and repair of mutational lesions (7). We are examining the regulation and function of a DNA methyltransferase, CcrM*, found in Caulobacter crescentus, a bacterium that undergoes cellular differentiation during each cell cycle (9). Caulobacter chromosomal DNA exhibits cell cycle-dependent patterns of methylation. Constitutive expression of the ccrM gene, yielding chromosomes that are fully methylated throughout the cell cycle, results in an altered developmental program, indicating that variations in methylation state are of regulatory significance (10). Understanding the role of DNA methylation in growth and development has been an elusive goal in many systems. There is abundant evidence correlating the level of cytosine methylation of eukaryotic DNA with gene expression and/or differentiation states (3)(4)(5)(11)(12)(13), but only recently has the role of DNA methylation in eukaryotic organisms been addressed genetically. A deficiency in cytosine methylation results in embryonic lethality in mice (14); in contrast, mutations resulting in deficiencies in DNA methylation in Arabidopsis thaliana (15, 16) and Neurospora crassa (17) are not lethal but cause abnormalities in chromosome segregation behavior. In prokaryotes, the only "regulatory" DNA methyltransferase that has been extensively examined has been the Dam methyltransferase of Escherichia coli and related enterics. Dam methylation is important for temporal control of chromosomal replication (1, 2) and for directing mismatch repair (7) The ccrM (cell-cycle regulated methyltransferase) locus encodes a DNA methyltransferase (CcrM) responsible for N6 methylation of adenine in GANTC sequences (10). In Caulobacter, the single chromosome replicates just once during the cell cycle (19,20). We have shown previously that the remethylation of newly replicated (and thereby hemimethylated) GANTC sites is restricted to the predivisional cell (10), near completion of chromosome replication, and that transcription of the ccrM gene occurs during a similar time fram...

CcrM, an adenine DNA methyltransferase, is essential for viability in Caulobacter crescentus. The CcrM protein is present only in the predivisional stage of the cell cycle, resulting in cell-cycle-dependent variation of the DNA methylation state of the chromosome. The availability of CcrM is controlled in two ways: (1) the ccrM gene is transcribed only in the predivisional cell, and (2) the CcrM protein is rapidly degraded prior to cell division. We demonstrate here that CcrM is an important target of the Lon protease pathway in C. crescentus. In a lon null mutant, ccrM transcription is still temporally regulated, but the CcrM protein is present throughout the cell cycle because of a dramatic increase in its stability that results in a fully methylated chromosome throughout the cell cycle. Because the Lon protease is present throughout the cell cycle, it is likely that the level of CcrM in the cell is controlled by a dynamic balance between temporally varied transcription and constitutive degradation. We have shown previously that restriction of CcrM to the C. crescentus predivisional cell is essential for normal morphogenesis and progression through the cell cycle. Comparison of the lon null mutant strain with a strain whose DNA remains fully methylated as a result of constitutive expression of ccrM suggests that the effect of Lon on DNA methylation contributes to several developmental defects observed in the lon mutant. These defects include a frequent failure to complete cell division and loss of precise cell-cycle control of initiation of DNA replication. Other developmental abnormalities exhibited by the lon null mutant, such as the formation of abnormally long stalks, appear to be unrelated to altered chromosome methylation state. The Lon protease thus exhibits pleiotropic effects in C. crescentus growth and development.

The Caulobacter crescentus DNA methyltransferase CcrM (M.CcrMI) methylates the adenine residue in the sequence GANTC. The CcrM DNA methyltransferase is essential for viability, but it does not appear to be part of a DNA restriction-modification system. CcrM homologs are widespread in the alpha subdivision of gramnegative bacteria. We have amplified and sequenced a 258-bp region of the ccrM gene from several of these bacteria, including Rhizobium meliloti, Brucella abortus, Agrobacterium tumefaciens, and Rhodobacter capsulatus. Alignment of the deduced amino acid sequences revealed that these proteins constitute a highly conserved DNA methyltransferase family. Isolation of the full-length ccrM genes from the aquatic bacterium C. crescentus, the soil bacterium R. meliloti, and the intracellular pathogen B. abortus showed that this sequence conservation extends over the entire protein. In at least two alpha subdivision bacteria, R. meliloti and C. crescentus, CcrMmediated methylation has important cellular functions. In both organisms, CcrM is essential for viability. Overexpression of CcrM in either bacterium results in defects in cell division and cell morphology and in the initiation of DNA replication. Finally, the C. crescentus and R. meliloti ccrM genes are functionally interchangeable, as the complemented strains are viable and the chromosomes are methylated. Thus, in both R. meliloti and C. crescentus, CcrM methylation is an integral component of the cell cycle. We speculate that CcrM-mediated DNA methylation is likely to have similar roles among alpha subdivision bacteria.DNA methylation has been identified in both prokaryotes and eukaryotes and has been implicated in many critical cellular processes, including transcriptional regulation (4, 40), initiation of DNA replication (3, 6, 28) and genomic imprinting (33, 34). In mammals and plants, DNA methylation is essential for development. Mice with a null mutation in a cytosine DNA methyltransferase die as embryos (19), and reduced cytosine methylation in Arabidopsis thaliana results in major defects in vegetative and reproductive development (12,35). In prokaryotes, DNA methylation is largely associated with DNA restriction-modification (R/M) systems, where its main function is to allow the cell to differentiate between self and foreign DNA (2, 41). However, bacterial DNA methylation does have other significant biological roles. In Escherichia coli, the Dam DNA methyltransferase (see reference 25 for a recent review), which is not part of an R/M system, regulates several cellular processes, including mismatch repair (15, 24), control of initiation of DNA replication (3,20), and the regulation of gene expression (30,40). Although Dam methylation is involved in a variety of important physiological functions, it is not essential for viability (23). The E. coli Dam methyltransferase is present in several related enteric bacteria, where its function is probably conserved (1).In the bacterium Caulobacter crescentus, the CcrM (M.CcrMI) DNA methyltransferase methyl...

The CcrM DNA methyltransferase of the ␣-proteobacteria catalyzes the methylation of the adenine in the sequence GAnTC. Like Dam in the enterobacteria, CcrM plays a regulatory role in Caulobacter crescentus and Rhizobium meliloti. CcrM is essential for viability in both of these organisms, and we show here that it is also essential in Brucella abortus. Further, increased copy number of the ccrM gene results in striking changes in B. abortus morphology, DNA replication, and growth in murine macrophages. We generated strains that carry ccrM either on a low-copy-number plasmid (strain GR131) or on a moderate-copy-number plasmid (strain GR132). Strain GR131 has wild-type morphology and chromosome number, as assessed by flow cytometry. In contrast, strain GR132 has abnormal branched morphology, suggesting aberrant cell division, and increased chromosome number. Although these strains exhibit different morphologies and DNA content, the replication of both strains in macrophages is attenuated. These data imply that the reduction in survival in host cells is not due solely to a cell division defect but is due to additional functions of CcrM. Because CcrM is essential in B. abortus and increased ccrM copy number attenuates survival in host cells, we propose that CcrM is an appropriate target for new antibiotics.Changes in DNA methylation patterns signal changes in cellular physiology in both prokaryotes and eukaryotes. In bacteria, DNA methyltransferases not only participate in restriction-modification systems (5) but also play regulatory roles in the cell. For example, methylation of the origin of replication by the Dam methyltransferase governs the timing of the initiation of DNA replication in Escherichia coli (6,29). Dam methylation also contributes to strand discrimination in methyl-directed DNA mismatch repair (19) and plays a role in pathogenesis (10, 12). For instance, changes in Dam methylation patterns control pyelonephritis-associated pilus (pap) transcription in uropathogenic E. coli by altering the binding of Lrp (leucine-responsive protein) to the pap promoter (21, 38). In addition, Dam methylation either directly or indirectly regulates the transcription of a number of genes in Salmonella enterica serovar Typhimurium that are induced following infection of the host (12), suggesting that DNA methylation plays a role in the virulence of this organism.The Dam methyltransferases of E. coli and other ␥-proteobacteria have a counterpart in ␣-proteobacteria, a DNA adenine methyltransferase called CcrM (for cell cycle-regulated methyltransferase). This enzyme was originally described in Caulobacter crescentus, a bacterium that is easily synchronized and therefore amenable to cell cycle studies (42). Both Dam and CcrM catalyze the transfer of the methyl group from S-adenosylmethionine to the adenine of their target DNA sequences. Both enzymes apparently lack cognate restriction enzymes and instead regulate cell cycle events. However, unlike Dam, CcrM is required for cell viability, and its activity is tightly regula...

The gene (PRAII) encoding a secreted aspartate proteinase of Candida albicans has been cloned and sequenced. The nucleotide and deduced amino acid sequences ofPRAII are 77 and 73% identical, respectively, with the reported sequences of PRAIO also cloned from C. albicans. Southern analyses indicated that the genome of each strain examined (ATCC 10231 and ATCC 10261) contains PRAIO and PRAII. Northern (RNA) analyses showed that PRAII was expressed at a much higher level than was PRA10 when secretion of the proteinase by strain ATCC 10261 was induced with albumin.The opportunistic pathogen Candida albicans secretes an aspartate proteinase (EC 3.4.23.6) Enzyme purification and protein analysis. Ten-liter cultures of strain ATCC 10261 were grown in a medium containing 0.2% yeast extract, 0.2% bovine serum albumin, and 2% (wt/vol) glucose (12 h at 30°C with aeration at 10 liters min-1 and a speed of 400 rpm). After cell harvesting, the medium was adjusted from pH 3.0 to pH 7.0 with 6 M NaOH and concentrated to 400 to 500 ml by ultrafiltration (10,000 molecular weight cutoff). After dialysis for 12 h against 10 mM sodium citrate buffer (pH 6.8), the concentrate was applied to a DEAE-Sephacel column (26 by 2.5 cm) equilibrated with the same buffer, and the column was washed