Noncanonical SMC protein in Mycobacterium smegmatis restricts maintenance of Mycobacterium fortuitum plasmids

Michael W. Panas, Paras Jain, Hui Yang, Shimontini Mitra, Debasis Biswas, Alice R ebecca Wattam, Norman L. Letvin, William R. Jacobs

Research output: Contribution to journalArticle

11 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)13264-13271
Number of pages8
JournalProceedings of the National Academy of Sciences of the United States of America
Volume111
Issue number37
DOIs
StatePublished - Sep 16 2014

Fingerprint

Mycobacterium fortuitum
Mycobacterium smegmatis
Plasmids
Chromosomes
Maintenance
Proteins
DNA
Phenotype
Amino Acid Sequence Homology
Mycobacterium leprae
Escherichia coli Proteins
Leprosy
Mycobacterium
Mycobacterium tuberculosis
Genes
Tuberculosis

Keywords

  • cell division
  • DNA topology
  • electroporation
  • partition errors
  • plasmid segregation

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Noncanonical SMC protein in Mycobacterium smegmatis restricts maintenance of Mycobacterium fortuitum plasmids. / Panas, Michael W.; Jain, Paras; Yang, Hui; Mitra, Shimontini; Biswas, Debasis; Wattam, Alice R ebecca; Letvin, Norman L.; Jacobs, William R.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 111, No. 37, 16.09.2014, p. 13264-13271.

Research output: Contribution to journalArticle

Panas, Michael W. ; Jain, Paras ; Yang, Hui ; Mitra, Shimontini ; Biswas, Debasis ; Wattam, Alice R ebecca ; Letvin, Norman L. ; Jacobs, William R. / Noncanonical SMC protein in Mycobacterium smegmatis restricts maintenance of Mycobacterium fortuitum plasmids. In: Proceedings of the National Academy of Sciences of the United States of America. 2014 ; Vol. 111, No. 37. pp. 13264-13271.
@article{e9c7874c377b4b22b34998c28cd57063,
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.",
keywords = "cell division, DNA topology, electroporation, partition errors, plasmid segregation",
author = "Panas, {Michael W.} and Paras Jain and Hui Yang and Shimontini Mitra and Debasis Biswas and Wattam, {Alice R ebecca} and Letvin, {Norman L.} and Jacobs, {William R.}",
year = "2014",
month = "9",
day = "16",
doi = "10.1073/pnas.1414207111",
language = "English (US)",
volume = "111",
pages = "13264--13271",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "37",

}

TY - JOUR

T1 - Noncanonical SMC protein in Mycobacterium smegmatis restricts maintenance of Mycobacterium fortuitum plasmids

AU - Panas, Michael W.

AU - Jain, Paras

AU - Yang, Hui

AU - Mitra, Shimontini

AU - Biswas, Debasis

AU - Wattam, Alice R ebecca

AU - Letvin, Norman L.

AU - Jacobs, William R.

PY - 2014/9/16

Y1 - 2014/9/16

N2 - 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.

AB - 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.

KW - cell division

KW - DNA topology

KW - electroporation

KW - partition errors

KW - plasmid segregation

UR - http://www.scopus.com/inward/record.url?scp=84964315712&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84964315712&partnerID=8YFLogxK

U2 - 10.1073/pnas.1414207111

DO - 10.1073/pnas.1414207111

M3 - Article

VL - 111

SP - 13264

EP - 13271

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 37

ER -