3.A.12 The Septal DNA Translocator (S-DNA-T) Family

During sporulation in Bacillus subtilis, the SpoIIIE protein is believed to form a translocation pore at the leading edge of the nearly closed septum. It then allows passage of the bacterial chromosome across the septum, functioning as a DNA exporter. Thus, it exports DNA from the cell compartment in which it is expressed (Sharp and Pogliano, 2002). When defective, due to mutations in the spoIIIE gene, only the leading portion of the chromosome (near OriC) is successfully translocated across the septum into the forespore compartment. The E. coliFtsK protein is homologous to SpoIIIE, and it can free chromosomes trapped in vegetative septa. It acts at the bacterial septum to couple chromosome segregation to cell division, pumping the DNA through the closing vegetative septum. It has also been shown to function as a DNA motor protein that activates chromosome dimer resolution (Aussel et al., 2002). Homologues of SpoIIIE are involved in cell to cell DNA transfer during plasmid conjugation.

SpoIIIE is expressed constitutively in vegetative B. subtilis cells, and its homologues are found in a wide variety of bacteria, most of which do not sporulate. These proteins may thus function generally as DNA translocation pores in vegetative prokaryotic septa, rather than just in sporulation septa. SpoIIIE plays other roles in membrane fusion during engulfment of the forespore by the mother cell. Marquis et al. (2008) have provided convincing evidence that one function of SpoIIIE is to strip off bound proteins such as RNA polymerase, transcription factors, remodeling proteins off the DNA so naked DNA enters the forespore. They propose that the translocation-stripping activity of SpoIIIE plays a key role in reprogramming developmental gene expression in the forespore.

SpoIIIE and its homologues actively drive DNA transport using a mechanism dependent on ATP hydrolysis. Thus, the carboxy terminal cytoplasmic part of SpoIIIE is a DNA-dependent ATPase. It can track along DNA in the presence of ATP. The amino terminal membrane-embedded part of SpoIIIE (residues 1-175 with four putative TMSs) localizes it to the division septum. During sporulation, SpoIIIE appears to act as a DNA pump that actively moves one of the two replicated chromosomes into the prespore. The presence of homologues in a broad range of bacteria suggests that this mechanism for active transport of DNA is widespread. Burton et al. (2007) demonstrated that the two arms of the chromosome are simultaneously pumped into the forespore. Up to 70 molecules of SpoIIIE are recruited to the site of DNA translocation and assemble into complexes that could contain 12 subunits. Fusion of the septal membranes during cytokinesis precedes DNA translocation. A model for DNA transport in which the transmembrane segments of FtsK/SpoIIIE form linked DNA-conducting channels across the two lipid bilayers of the septum has been proposed (Burton et al., 2007).

SpoIIIE exports DNA with translocation polarity that is governed by the cell-specific regulation of its assembly, but FtsK is a reversible motor for which translocation polarity is governed by its DNA substrate. In differentiating B. subtilis cells, SpoIIIE assembles a complex only in the mother cell, from which DNA is exported. Becker and Pogliano (2007) have shown that altering chromosome architecture by soj-spo0J and racA soj-spo0j mutations allows wild-type SpoIIIE to assemble in the forespore and export the forespore chromosome. The chromosome is exported from the forespore when oriC fails to be trapped in the forespore. Thus, the position of oriC after septation determines which cell will receive the chromosome and which will assemble SpoIIIE.

A single plasmid-encoded protein, the septal DNA translocator TraB, is sufficient to promote conjugal plasmid transfer in mycelial streptomycetes. TraB proteins from plasmids pSG5 of Streptomyces ghanaensis and pSVH1 of Streptomyces venezuelae were characterized. TraB of pSG5 was expressed as a fusion protein with eGFP and found to be localized at the hyphal tips, which indicates that conjugation takes place at the tips of the mating mycelium. The TraB protein of pSVH1 was heterologously expressed with an N-terminal strep-tagII and purified as a soluble protein to near homogeneity. The purified protein was shown to hydrolyse ATP and to bind to a 50 bp non-coding pSVH1 sequence containing a 14 bp direct repeat. The protein-DNA complex was too large to enter an agarose gel, indicating that multimers of TraB were bound to the DNA. Denaturation of the protein-DNA complex released unprocessed plasmid DNA demonstrating that the TraB protein does not possess nicking activity. Thus, conjugal DNA transfer in streptomycetes is mediated by the septal DNA translocator TraB, a plasmid-encoded ATPase that interacts non-covalently with DNA and translocates an unprocessed double-stranded DNA molecule at the hyphal tip into the recipient.

The generalized reaction catalyzed by proteins of the S-DNA-T family may therefore be:

DNA or DNA-protein complex (in one cytoplasmic compartment) + nATP

DNA or DNA-protein complex (in an adjacent cytoplasmic compartment) + nADP + nPi.


 

References:

Aussel, L., F.X. Barre, M. Aroyo, A. Stasiak, A.Z. Stasiak, and D. Sherratt. (2002). FtsK is a DNA motor protein that activates chromosome dimer resolution by switching the catalytic state of the XerC and XerD recombinases. Cell 108: 195-205.

Bath, J., L.J. Wu, J. Errington, and J.C. Wang. (2000). Role of Bacillus subtilisSpoIIIE in DNA transport across the mother cell-prespore division septum. Science 290: 995-997.

Becker, E.C., and K. Pogliano. (2007). Cell-specific SpoIIIE assembly and DNA translocation polarity are dictated by chromosome orientation. Mol. Microbiol. 66: 1066-1079.

Berezuk, A.M., M. Goodyear, and C.M. Khursigara. (2014). Site-directed Fluorescence Labeling Reveals a Revised N-terminal Membrane Topology and Functional Periplasmic Residues in the Escherichia coli Cell Division Protein FtsK. J. Biol. Chem. 289: 23287-23301.

Biller, S.J. and W.F. Burkholder. (2009). The Bacillus subtilis SftA (YtpS) and SpoIIIE DNA translocases play distinct roles in growing cells to ensure faithful chromosome partitioning. Mol. Microbiol. 74: 790-809.

Burton, B.M., K.A. Marquis, N.L. Sullivan, T.A. Rapoport, and D.Z. Rudner. (2007). The ATPase SpoIIIE transports DNA across fused septal membranes during sporulation in Bacillus subtilis. Cell 131: 1301-1312.

Bush, M.J., N. Tschowri, S. Schlimpert, K. Flärdh, and M.J. Buttner. (2015). c-di-GMP signalling and the regulation of developmental transitions in streptomycetes. Nat. Rev. Microbiol. 13: 749-760.

Dubarry, N. and F.X. Barre. (2010). Fully efficient chromosome dimer resolution in Escherichia coli cells lacking the integral membrane domain of FtsK. EMBO. J. 29: 597-605.

Errington, J. (1998). Dramatic new view of bacterial chromosome segregation. ASM News 64: 210-217.

Fleming, T.C., J.Y. Shin, S.H. Lee, E. Becker, K.C. Huang, C. Bustamante, and K. Pogliano. (2010). Dynamic SpoIIIE assembly mediates septal membrane fission during Bacillus subtilis sporulation. Genes Dev. 24: 1160-1172.

Higgins, D. and J. Dworkin. (2012). Recent progress in Bacillus subtilis sporulation. FEMS Microbiol. Rev. 36: 131-148.

Kaimer, C., J.E. González-Pastor, and P.L. Graumann. (2009). SpoIIIE and a novel type of DNA translocase, SftA, couple chromosome segregation with cell division in Bacillus subtilis. Mol. Microbiol. 74: 810-825.

Marquis, K.A., B.M. Burton, M. Nollmann, J.L. Ptacin, C. Bustamante, S. Ben-Yehuda, and D.Z. Rudner. (2008). SpoIIIE strips proteins off the DNA during chromosome translocation. Genes Dev. 22: 1786-1795.

Pogliano, J., M.D. Sharp, and K. Pogliano. (2002). Partitioning of chromosomal DNA during establishment of cellular asymmetry in Bacillus subtilis. J. Bacteriol. 184: 1743-1749.

Reuther J., C. Gekeler, Y. Tiffert, W. Wohlleben, G. Muth. (2006). Unique conjugation mechanism in mycelial streptomycetes: a DNA-binding ATPase translocates unprocessed plasmid DNA at the hyphal tip. Mol. Microbiol. 61: 436-446.

Sharp, M.D. and K. Pogliano. (2002). Role of cell-specific SpoIIIE assembly in polarity of DNA transfer. Science 295: 137-139.

Sharp, M.D. and K. Pogliano. (1999). An in vivomembrane fusion assay implicates SpoIIIE in the final stages of engulfment during Bacillus subtilis sporulation. Proc. Natl. Acad. Sci. USA 96:14553-14558.

Sharpe, M.E. and J. Errington. (1999). Upheaval in the bacterial nucleoid: an active chromosome segregation mechanism. Trends Genet. 15: 70-74.

Sharpe, M.E., P.M. Hauser, R.G. Sharpe, and J. Errington. (1998). Bacillus subtilis cell cycle as studied by fluorescence microscopy: constancy of cell length at initiation of DNA replication and evidence for active nucleoid partitioning. J. Bacteriol. 180: 547-555.

Wu, L.J. and J. Errington. (1994). Bacillus subtilisSpoIIIE protein required for DNA segregation during asymmetric cell division. Science 264: 572-575.

Wu, L.J., P.J. Lewis, R. Allmansberger, P.M. Hauser, and J. Errington. (1995). A conjugation-like mechanism for prespore chromosome segregation during sporulation in Bacillus subtilis. Genes Dev. 9: 1316-1326.

Examples:

TC#NameOrganismal TypeExample
3.A.12.1.1

Septum DNA translocation pore protein SpoIIIE. SpoIIIE forms DNA-conducting channels across the two lipid bilayers of the septum (Burton et al., 2007). The dynamic assembly of SpoIIIE mediates septal membrane fission during Bacillus subtilis sporulation (Fleming et al., 2010).  Directionality of DNA pumping is probably a result of asymmetic chromosomal distrbution of SpoIIIE Recognition Sequences in the DNA.  Since SpoIIIE can strip proteins off the chromosome during translocation, SpoIIIE may interact with the DNA beyond it's binding sites (Higgins and Dworkin 2012).

Bacteria

SpoIIIE of Bacillus subtilis

 
3.A.12.1.2

Cell division protein, FtsK. FtsK lacking its integral membrane domain catalyzes fully efficient chromosome dimer resolution (Dubarry and Barre, 2010).  Its topology has been examined (Berezuk et al. 2014).

Bacteria

FtsK of E. coli

 
3.A.12.1.3

SpoIIIE/FtsK homologue of 948 aas

Spirochaetes

FtsK homologue of Leptospira interrogans

 
3.A.12.1.4

Cell division DNA translocase, FtsK of 917 aas and 4 TMSs (Bush et al. 2015).

FtsK of Streptomyces coelicolor

 
3.A.12.1.5

DNA translocase, SftA (YtpS; YtpT) of 952 aas.  Required for the accurate completion of chromosome partitioning, in part by promoting efficient resolution of chromosome dimers, before the formation of the division septum. Binds to DNA in a non-specific manner. Shows ATPase activity. Not required for cytokinesis (Biller and Burkholder 2009; Kaimer et al. 2009).

SftA of Bacillus subtilis

 
Examples:

TC#NameOrganismal TypeExample
3.A.12.2.1The major protein required for plasmid transfer, TraBacteriaTra of Streptomyces lividans
 
Examples:

TC#NameOrganismal TypeExample
3.A.12.3.1The DNA-binding translocating ATPase, TraB (translocates unprocessed plasmid DNA at the hyphal tip in a conjugation-like process) (Reuther et al, 2006). Bacteria TraB of plasmid pSG5 of Streptomyces ghanaensis
(O70031)
 
3.A.12.3.2The DNA-binding translocating ATPase, TraB (translocates unprocessed plasmid DNA at the hyphal tip in a conjugation-like process) (Reuther et al, 2006). BacteriaTraB of plasmid pSV of Streptomyces ghanaensis
(Q3LAJ2)