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

Abstract

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.

FIG. 1

The A. tumefaciens ccrM homolog is essential for viability. (A) Homology diagram of the A. tumefaciens (Atu) CcrM homolog in relation to the other homologs from S. meliloti (Sme), B. abortus (Bab), and C. crescentus (Ccr). Black boxes denote identical amino acids and shaded boxes denote similar amino acids. The catalytic and S-adenosylmethionine-binding domains are marked. (B) (Top) Diagram of the ccrM locus. (Bottom) Diagram of the locus following integration of the plasmid pLK311, yielding tandem copies of the gene, one of which is disrupted by a spectinomycin resistance cassette. The resulting strain is resistant to both kanamycin and spectinomycin. Growth on 5% sucrose with excision of the vector sequences leads to two possible outcomes. If excision occurs on the opposite side of the Ω cassette from the original integration event, a disrupted copy of the gene will remain on the chromosome and the resulting strain will be spectinomycin resistant but kanamycin sensitive. If excision occurs on the same side as the original integration event, then the strain will be wild type and sensitive to both spectinomycin and kanamycin. (C) Only the presence of ccrM in trans allows viable colonies when the chromosomal copy of ccrM is disrupted. Strain LS3307 carries the pLK311 vector recombined on the 5′ side of the spectinomycin resistance cassette, and LS3308 carries pLK311 recombined on the 3′ side of the cassette. Results are shown for both strains with no plasmid, vector alone [pRK290(20R)], or vector plus ccrM (pLK308). A minimum of 100 isolates were scored for each experiment.

FIG. 2

Overexpression of the ccrM homolog leads to an increase in the methylation of chromosomal DNA. (A) Schematic of the DNA methylation assay. CcrM methylates adenine in the sequence GANTC (shaded). This CcrM site overlaps the HincII restriction enzyme recognition site GTTGAC in the att locus. If the DNA is fully methylated by CcrM, HincII will not be able to cleave it. However, once this DNA is replicated, each replication product will be hemimethylated, as shown below. The top strand is methylated in one product (left); the bottom strand is methylated in the other product (right). Only one of these two replication products can be cleaved by HincII, as shown. Southern blotting with the probe shown will yield a 1.3-kb band or a 1-kb band, depending on the methylation state of the DNA. (B) Methylation state of the A. tumefaciens A348 DNA in strains carrying different plasmids, using the differential HincII cleavage assay described for panel A. Genomic DNA from mixed cultures in exponential-phase growth was digested with HincII. The low-copy-number vector is pRK290(20R); the low-copy-number vector with ccrM is pLK308 [pRK290(20R) with ccrM]; the high-copy-number vector is pJS71; the high-copy-number vector with ccrM is pLK309 (pJS71 with ccrM). The upper band contains fully methylated (FM) DNA and the hemimethylated (HM) DNA diagrammed on the left in panel A, while the lower band contains the hemimethylated DNA diagrammed on the right. Shown directly below is the ratio between bands for each lane of the gel. (C) Light microscopy of A. tumefaciens A348 carrying different plasmids, which are detailed for panel B above. Bar, 5 μm. (D) Flow cytometry of the strains in panel C. The y axis represents the number of cells with a given fluorescence intensity. The x axis shows the fluorescence intensity, which corresponds to DNA content.

FIG. 3

The A. tumefaciens ccrM promoter has a highly conserved sequence and is bound by the CtrA response regulator. (A) Sequence of the ccrM promoter region and its alignment with the ccrM promoters of C. crescentus and B. abortus. The conserved CtrA binding site (consensus, TTAANTTAAC) is in bold type. The region protected from DNase I by phosphorylated CtrA is shaded. (B) Start site of ccrM transcription as determined by primer extension. A sequence ladder generated with the same primer was used to determine the start site. (C) DNase I protection of the ccrM promoter by CtrA. A sequence ladder generated by using the primer AgMTFP1 was used to identify the protected region. Protection was seen only if CtrA was phosphorylated by the histidine kinase EnvZ.

FIG. 4

A. tumefaciens A348 can be synchronized, and the methylation state of the chromosome varies as a function of the cell cycle. (A) Cells were separated using density centrifugation, as described in Materials and Methods. Flow cytometry of the cells isolated from the bottom and the top of the gradient is shown. (B) The cells from the bottom of the gradient were isolated and allowed to grow as described in Materials and Methods. The changes in motility are indicated; DNA content, as assessed by flow cytometry, and cell morphology over time are shown. The assessment of motility reflects the behavior of the majority of the cell population, as observed by light microscopy, at the time the sample was taken. A vertical line has been arbitrarily placed at 75 fluorescence units on the x axis to aid in following the relative positions of the peaks over the course of the experiment. Bar, 5 μm. (C) Western blot of A. tumefaciens flagellin proteins using polyclonal antiserum to Caulobacter flagellins. The specific 32- to 33-kDa band is shown. Mixed cell populations from both wild-type (A348) and flagellin-negative (NT1REB) cells are shown in the first and second lanes as positive and negative controls. The remaining lanes track the flagellins over the course of the cell cycle in A348. (D) Genomic DNA was prepared from cells at the time points shown in panel B, and the methylation state of the att locus was assayed as described in Materials and Methods and for Fig. 2. A ratio of one between the bottom band and the top band corresponds to 100% hemimethylation of the DNA.