Membrane-associated DNA transport machines

Abstract

DNA pumps play important roles in bacteria during cell division and during the transfer of genetic material by conjugation and transformation. The FtsK/SpoIIIE proteins carry out the translocation of double-stranded DNA to ensure complete chromosome segregation during cell division. In contrast, the complex molecular machines that mediate conjugation and genetic transformation drive the transport of single stranded DNA. The transformation machine also processes this internalized DNA and mediates its recombination with the resident chromosome during and after uptake, whereas the conjugation apparatus processes DNA before transfer. This article reviews these three types of DNA pumps, with attention to what is understood of their molecular mechanisms, their energetics and their cellular localizations.

Figure 1.

Localization of chromosome segregation proteins, SftA and SpoIIIE, in Bacillus subtilis. (A) SftA localizes to early division septa in B. subtilis, whereas SpoIIIE localizes late in division and largely sporulation septa. This image shows fluorescence overlayed from FM4-64 membrane-staining (red), a SftA-YFP fusion (green) and a SpoIIIE-CFP fusion (blue), of cells from a sporulating culture. SftA fluorescence is apparent at predivisional and vegetative divsion sites, whereas SpoIIIE fluorescence colocalizes with fully formed sporulation division membranes. (Images courtesy of J. Liu.) (B) Schematic of the chromosome crossing the division septum through two independent DNA-conducting channels formed by SpoIIIE in a sporulating B. subtilis cell. At the end of the transport process, the final loop of DNA in the large compartment must be resolved across the division plane, by an as yet undetermined mechanism.

Figure 2.

Proposed structure of the core complex for T4S system (pKM101)-mediated conjugation. The cryo-EM structure (yellow) is superimposed on the crystal structure of the outer-membrane complex. The location of lipid associated with TraN/VirB7 is shown with black dots. The arrows illustrate proposed conformational changes (Chandran et al. 2009). OM and IM indicate outer and inner membrane, respectively. (Adapted, with permission, from Chandran et al. 2009 [© Nature Publishing Group].)

Figure 3.

A model for transformation in Gram positive bacteria. ComGC is processed by the protease ComC and oxidized to form an intramolecular disulfide bond by BdbDC. (A) It is then translocated from the membrane in a step that requires the traffic ATPase ComGA and assembled into a pseudopilus in which the intramolecular disulfide bonds are replaced by links between the subunits. ComGB is represented at the base of the pseudopilus without direct evidence. It is proposed that assembly and disassembly of the pseudopilus brings transforming DNA to the ComEC channel and that interaction of the DNA with the C-terminal part of ComEA is required for uptake, which is assisted by the ATPase ComFA. The transforming strand (gray) enters the cytosol and the nontransforming strand is degraded with the products exiting the cell. The nomenclature is that of B. subtilis. In S. pneumoniae, EndA (not shown) carries out degradation of the non-transforming strand. Also not shown is NucA, which introduces double strand breaks in the bound DNA. The large gray square represents cell wall material. (B) The pseudopilus has disassembled and the ComGC subunits have returned to the membrane where they may form a pool available for further cycles of assembly/disassembly. As the ssDNA enters the cytosol, it interacts with SsbB and DprA. These proteins protect the DNA and DprA also assists in loading RecA to form a filament, which is proposed to carry out the homology search leading to recombination with the resident chromosome

Figure 4.

ComGA-GFP localizes to the poles of competent B. subtilis. This image shows fluorescence from DAPI-staining (blue), and a ComGA-GFP fusion (green), overlayed on a DIC image of ten cells from a competent culture. Two competent cells are present, in both of which the ComGA fluorescence is localized near the cell poles. (Photo courtesy of J. Hahn.)