Recruitment, assembly, and molecular architecture of the SpoIIIE DNA pump revealed by superresolution microscopy

PLoS Biol. 2013;11(5):e1001557. doi: 10.1371/journal.pbio.1001557. Epub 2013 May 7.

Abstract

ATP-fuelled molecular motors are responsible for rapid and specific transfer of double-stranded DNA during several fundamental processes, such as cell division, sporulation, bacterial conjugation, and viral DNA transport. A dramatic example of intercompartmental DNA transfer occurs during sporulation in Bacillus subtilis, in which two-thirds of a chromosome is transported across a division septum by the SpoIIIE ATPase. Here, we use photo-activated localization microscopy, structured illumination microscopy, and fluorescence fluctuation microscopy to investigate the mechanism of recruitment and assembly of the SpoIIIE pump and the molecular architecture of the DNA translocation complex. We find that SpoIIIE assembles into ∼45 nm complexes that are recruited to nascent sites of septation, and are subsequently escorted by the constriction machinery to the center of sporulation and division septa. SpoIIIE complexes contain 47±20 SpoIIIE molecules, a majority of which are assembled into hexamers. Finally, we show that directional DNA translocation leads to the establishment of a compartment-specific, asymmetric complex that exports DNA. Our data are inconsistent with the notion that SpoIIIE forms paired DNA conducting channels across fused membranes. Rather, our results support a model in which DNA translocation occurs through an aqueous DNA-conducting pore that could be structurally maintained by the divisional machinery, with SpoIIIE acting as a checkpoint preventing membrane fusion until completion of chromosome segregation. Our findings and proposed mechanism, and our unique combination of innovating methodologies, are relevant to the understanding of bacterial cell division, and may illuminate the mechanisms of other complex machineries involved in DNA conjugation and protein transport across membranes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus subtilis / genetics*
  • Bacillus subtilis / metabolism*
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Conjugation, Genetic
  • DNA, Bacterial / genetics
  • DNA, Bacterial / metabolism
  • Microscopy, Fluorescence
  • Spores, Bacterial / metabolism

Substances

  • Bacterial Proteins
  • DNA, Bacterial
  • spore-specific proteins, Bacillus

Grant support

Financial support was provided by the Human Frontiers Science Program (CDA-00017/2009, MN) and the Institut National de la Santé et la Recherche Médicale (Avenir Programme, MN). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.