Transport of DNA across the outer membrane presents two big problems, neither of which have been well articulated to date. The first is the difficulty of getting started, which requires pulling an initial loop of double stranded DNA across the membrane. The second is the difficulty of continuing to pull a long fiber of DNA into the confined space of the periplasm.
Both are probably explained by the ability of the fibers called type IV pili to bind to DNA and to be pulled into the periplasm. Type IV pili are long narrow threads composed of identical protein subunits (pilins) arranged in a rope-like helical coil (abut 6nm wide and up to 5 µm long). They are assembled in the periplasm by adding subunits to the base, and elongated and retracted by addition and removal of subunits. The pili that pull DNA are really 'pseudopili' because they're so short, but for simplicity I'll refer to both long and short ones as pili.
I've posted before about the getting-started problem (see for example Getting the kinks in), so here I'll try to explain the continuing-to-pull problem, whose importance I only realized a few days ago while discussing DNA uptake with one of the post-docs. Let's assume that a loop of DNA has bound to the pilus and been pulled across the outer membrane into the periplasm. And for now let's assume that we need only consider what's happening to the DNA of one side of the loop, and can ignore what might be happening to the other side of the loop.
The big problem is that retraction of the pilus can only pull the DNA in a short ways, no more than the length of the pilus. As the pili that transport DNA are too short to see even with electron microscopy, we think they must be barely long enough to protrude through the secretin pore, probably less than 20nm long. This is only 5 turns of the pilin helix, and only about 20bp of DNA. So how does the pilus pull in DNA molecules that are at least 10 kb long? Here are figures illustrating two solutions.
The first is compatible with the way long pili cause adhesion and 'twitching motility'. The pilus attaches to DNA, pulls it in a bit by disassembling pilin subunits at the base, pulling the pilus down. It then lets go of the DNA, elongates a bit by adding subunits at the base, and grabs a fresh part of the DNA to pull on. In the figure I've shown the pilus as short enough to not protrude through the secretin pore, leaving the pore free for the DNA. This would limit the length of each pilus 'stroke' to the thickness of the periplasm. Under normal circumstances this would be only about 10nm, but the pilus may push the membranes further apart.
The second model is more elegant, but requires a new mechanism that we have no direct evidence for. The first two steps are the same; the pilus binds DNA and pulls it in by disassembling subunits at the base of the pilus, which pulls the pilus down. But in this model new subunits are continuously added to the other end of the pilus, so it never gets shorter and continuously binds and pulls new DNA down into the periplasm.
The drawings are more-or-less to scale, but the periplasmic space may be thicker than shown, which would allow the pilus to be longer without obstructing the pore.
The first model is more parsimonious, in that it uses the retraction mechanism that we know works for the long pili that mediate adhesion and twitching motility. Initially I thought that the pilus might have difficulty letting go of the DNA before elongating, but when I drew the model I realized that, once the pilus becomes very short it will be unable to bind DNA and so release will be spontaneous.
Can we design experiments that distinguish between these models? And is this something that our big grant proposal should address? I'll leave the answers for another post.
11 hours ago in Variety of Life