Sub­tle Bacil­lus sub­tilis

by Elio

When Bacil­lus sub­tilis sporu­lates, the cell des­tined to make a spore di­vides asym­met­ri­cally; one cell, the "fore­spore," be­comes the spore; the other, a short-lived sa­crificial "mother cell." You'd ex­pect that a nu­cleoid would be par­ti­tioned be­tween these two com­part­ments be­fore they even think of di­vid­ing. Not so. Cell di­vi­sion and chro­mosome seg­re­ga­tion get out of sync and the sep­tum be­tween the two cells nar­rows be­fore the DNA is complete­ly trans­ferred into the fu­ture spore. Never mind why this is so – the rea­sons are elu­sive. Re­gard­less, about 2/3 of the DNA des­tined for the spore has to tra­verse a nar­row pore in the fore­s­pore sep­tum. A pro­tein called SpoI­IIE (names like this one mean some­thing to sporu­la­tion mavens) is deeply in­volved and acts as a DNA translo­case.

Spore for­ma­tion in B. sub­tilis be­gins with an asym­metric di­vi­sion gen­er­at­ing a lar­ge mother cell and a small fore­s­pore cell. Ini­tially, these two cells lie side-by-side but later in de­vel­op­ment the mother cell en­gulfs the fore­s­pore in a phago­cytic-like process to pro­duce a cell-within-a-cell. Pic­­tures by D. Rud­ner. Source

How does SpoI­IIE know to push (or pull) one way and not the other? Does it rec­og­nize se­quences in the DNA (model A), thus be­ing po­ten­tially able to move DNA across the sep­tum in ei­ther direc­tion? Or is it part of a com­plex that acts as a one-di­rec­tional winch (model B)? In model B, the di­rectionality would set by as­sem­bling the winch on just one side of the sep­tum (see fig­ure be­low).

A If SpoIIIE–Jun^LZ (white cir­cles) as­sem­bles only in the mother cell, Fos^LZ –GFP (green) will form a sep­tal fo­cus when ex­pressed in the mother cell, but not when ex­pressed in the fore­s­pore. B If it as­sem­bles in both cells, Fos^LZ –GFP (green) will form a fo­cus when ex­pressed in ei­ther cell. Source

Eric Becker and Kit Pogliano dreamt up a way of an­swer­ing this ques­tion. How they did it is mighty clever (and some­what in­tri­cate). To de­ter­mine the po­si­tion of SpoI­IIE rel­a­tive to the sep­tum, they fused the leucine zip­per do­main (LZ) of cFos to SpoI­IIE and fused the LZ of cJun to GFP (green flu­o­res­cent pro­tein). cFos and cJun are bio­logically ir­rel­e­vant pro­teins here, but they are used be­cause their two LZ do­mains strongly in­ter­act with each other. Us­ing cell-spe­cific pro­mot­ers, syn­the­sis of cJun-GFP can be turned on in ei­ther the mother cell or the spore com­part­ment. Ergo, they could lo­cal­ize SpoI­IIE to one or the other site.

They got a clear an­swer: SpoI­IIE is as­sem­bled only on the mother cell side of the sep­tum (as pre­dicted by Model B). Thus it seems to push the DNA rather than pull it across.

Fos^LZ-GFP forms a fo­cus at the sep­tum only when ex­pressed in the mother cell (C, ar­rows), not when ex­pressed in the fo­respore (D, ar­row­heads). Red: mem­branes stained with FM4-64. Green: DNA stained with CY­TOX-green. Source

But there is an­other side to the story. If DNA transloca­tion is im­paired, the pro­tein lo­cal­izes at both sides of the sep­tum, which sug­gests that the translo­cated DNA has a say in the mat­ter (model A). In fur­ther ex­per­i­ments, they con­firmed the par­tic­i­pa­tion of the DNA by show­ing that the ori­en­ta­tion of the chro­mo­some also counts. Spe­cific mo­tifs on the DNA per­mit SpoI­IIE to winch it in one di­rect­ion only.

This work is an el­e­gant mix­ture of clever ge­netic manipu­lation and mouth-wa­ter­ing flu­o­res­cence mi­croscopy. In sum, their re­sults con­firm the no­tion that prop­er­ties of both the SpoI­IIE as­sem­bly and the DNA par­tic­i­pate in proper translo­ca­tion. Mov­ing DNA around a sporu­lat­ing cell is not a game for be­gin­ners. It takes mol­e­c­u­lar so­phistication to do it right.

Note: The species name sub­tilis comes from the Latin for "fine, thin, del­i­cate." In 14th cen­tury French, it mor­phed into sub­til, mean­ing "clever, dex­ter­ous." We think that the evo­lu­tion of the word mim­ics the evo­lu­tion of the or­gan­ism. Nomen est omen.