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...
To determine the placental tropism and abortigenicity of the vaccine candidate Brucella abortus strain RB51 (SRB51), a rough mutant of the virulent strain 2308, ten Polled Hereford heifers were inoculated intravenously in the 6th month of gestation. Heifers were euthanatized and examined at postinoculation week (PIW) 8 (n = 5) or at full term (n = 5). Four of five infected heifers sampled at PIW 8 and three of four infected heifers at term had placentitis, whereas reproductive tissues of three normal cows used for comparison had no placentitis. Numerous macrophages, immunoreactive for SRB51 antigen, as well as neutrophils, fibrin, and cell debris filled the arcade zone between chorion and maternal septae. Trophoblastic epithelium of the placentomal arcade zone had intracellular bacteria that were immunoreactive for SRB51 antigen. The tips of maternal septa had a lymphoplasmacytic infiltrate with small multifocal erosions and ulcerations of maternal epithelium. SRB51 was cultured from all tissues in which lesions were seen. Placentae of one cow from each group had no placentitis and contained no SRB51. In mammae, interstitial lymphoplasmacytic infiltrates and suppurative infiltrates within alveoli and intralobular ductules were seen in two of five heifers at PIW 8. SRB51 was cultured from liver, spleen, lung, and bronchial lymph nodes in four of five calves at PIW 8 and three of four full-term calves, but no lesions were seen. One near-term heifer had disseminated infection, placentitis, and lymphoplasmacytic endometritis, and delivered a premature weak calf. These results establish that SRB51 is less abortifacient than previously published reports with strain 19, in that only one of four heifers delivered prematurely following intravenous inoculation with SRB51, whereas intravenous inoculation with strain 19 leads to 100% abortion. However, it also shows that SRB51 can infect the bovine placenta, mammary gland, and fetus, can induce placentitis, and, in some cases, can lead to preterm expulsion of the fetus.
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