title: Noncanonical SMC protein in Mycobacterium smegmatis restricts maintenance of Mycobacterium fortuitum plasmids.
abstract: Research on tuberculosis and leprosy was revolutionized by the development of a plasmid transformation system in the fast-growing surrogate, Mycobacterium smegmatis. This transformation system was made possible by the successful isolation of a M. smegmatis mutant strain mc(2)155, whose efficient plasmid transformation (ept) phenotype supported the replication of Mycobacterium fortuitum pAL5000 plasmids. In this report, we identified the EptC gene, the loss of which confers the ept phenotype. EptC shares significant amino acid sequence homology and domain structure with the MukB protein of Escherichia coli, a structural maintenance of chromosomes (SMC) protein. Surprisingly, M. smegmatis has three paralogs of SMC proteins: EptC and MSMEG_0370 both share homology with Gram-negative bacterial MukB; and MSMEG_2423 shares homology with Gram-positive bacterial SMCs, including the single SMC protein predicted for Mycobacterium tuberculosis and Mycobacterium leprae. Purified EptC was shown to bind ssDNA and stabilize negative supercoils in plasmid DNA. Moreover, an EptC-mCherry fusion protein was constructed and shown to bind to DNA in live mycobacteria, and to prevent segregation of plasmid DNA to daughter cells. To our knowledge, this is the first report of impaired plasmid maintenance caused by a SMC homolog, which has been canonically known to assist the segregation of genetic materials.
authors.affiliation: Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115;
authors.forename: Michael W
authors.initials: MW
authors.lastname: Panas
authors.affiliation: Howard Hughes Medical Institute and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461;
authors.forename: Paras
authors.initials: P
authors.lastname: Jain
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461;
authors.forename: Hui
authors.initials: H
authors.lastname: Yang
authors.affiliation: Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115;
authors.forename: Shimontini
authors.initials: S
authors.lastname: Mitra
authors.affiliation: Department of Microbiology, Himalayan Institute of Medical Sciences, Swami Ram Nagar, Jolly Grant, Dehradun 248140, India; and.
authors.forename: Debasis
authors.initials: D
authors.lastname: Biswas
authors.affiliation: Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA 24061.
authors.forename: Alice Rebecca
authors.initials: AR
authors.lastname: Wattam
authors.affiliation: Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115;
authors.forename: Norman L
authors.initials: NL
authors.lastname: Letvin
authors.affiliation: Howard Hughes Medical Institute and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461; william.jacobs@einstein.yu.edu.
authors.forename: William R
authors.initials: WR
authors.lastname: Jacobs
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D019295:Computational Biology; D017353:Gene Deletion; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019907:Mycobacterium fortuitum / Q000378:metabolism*; D020102:Mycobacterium smegmatis / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014170:Transformation, Genetic
chemical_list: D001426:Bacterial Proteins
keywords: DNA topology; cell division; electroporation; partition errors; plasmid segregation