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Journal of Bacteriology, June 2000, p. 3482-3489, Vol. 182, No. 12
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

The Brucella abortus CcrM DNA Methyltransferase Is Essential for Viability, and Its Overexpression Attenuates Intracellular Replication in Murine Macrophages

Gregory T. Robertson,1,dagger Ann Reisenauer,2 Rachel Wright,2,Dagger Rasmus B. Jensen,2 Allen Jensen,3 Lucille Shapiro,2 and R. Martin Roop II1,*

Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 711301; Department of Developmental Biology, Stanford University School of Medicine, Stanford, California 943052; and National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Ames, Iowa 500103

Received 4 February 2000/Accepted 21 March 2000

The CcrM DNA methyltransferase of the alpha -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.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, P.O. Box 33932, Louisiana State University Health Science Center, 1501 Kings Highway, Shreveport, LA 71130-3932. Phone: (318) 675-5771. Fax: (318) 675-5764. E-mail: rroop{at}lsumc.edu.

dagger Present address: Lilly Research Laboratories, Eli Lilly & Company, Indianapolis, IN 46285.

Dagger Present address: Incyte Pharmaceuticals, Palo Alto, CA 94304.

Journal of Bacteriology, June 2000, p. 3482-3489, Vol. 182, No. 12
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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