The old uptake-specificity papers I'm reading (to reanalyze their uptake data, see previous post) include gels showing pools of radioactively-labeled DNA fragments before and after being taken up by cells. In addition to the data I'm looking for, these gels remind me of a puzzle involving a gene for a ligase.
before | uptake
Usually the 'before' pool used in these gels contains end-labeled restriction fragments of a plasmid containing a H. influenzae DNA insert. All the bands are of equal intensity because there are the same number of molecules of each piece of the plasmid. The left lane of the gel figure shows such a 'before' sample. The labeled DNA is then mixed with competent cells, and after a few minutes the DNA that hasn't been taken up is washed away and/or degraded with DNaseI, and DNA is purified from the cells and run in a gel with the 'before' sample. (This 'uptake' DNA prep includes the cells' own DNA, but that's not radioactive so it doesn't show up on the gel.)
There are two kinds of differences between the before and after lanes. First, some of the 'before' bands are missing (or faint) in the 'uptake' lane. That's because these fragments lack USSs and so were taken up poorly or not at all. Second, faint new bands appear that weren't in the Before sample. These are due to the ligase puzzle.
The authors of the paper said that the new bands appeared because of a ligase enzyme that joins the ends of DNA fragments while they are in the periplasm (the space between the outer and inner membranes. Similar bands were seen in similar experiments in later papers from this lab. A ligase in the periplasm has also been invoked to explain the presence of joined-together DNA fragments recombined into chromosomal DNA.
But the whole idea of a periplasmic ligase seemed a bit odd, as what would a ligase do in the periplasm? There isn't normally any DNA there, and even during DNA uptake there's no obvious role for a ligase.
However, when we did a microarray analysis to identify the genes turned on when cells become competent (see link to CRE-2005 paper in sidebar), we found that one of the genes turned on encodes a ligase with a signal sequence that should target it to the periplasm. Unbeknownst to us, the enzyme had already been well-characterized by biochemists - it's a real ligase, but the ATP-dependent kind typical of some phages, rather than the NAD-dependent kind that acts in DNA replication and repair.
So not only were the early researchers correct in invoking a ligase in the periplasm, but this ligase is specifically turned on when cells are preparing to take up DNA. Consistent with such a role, VanWagoner knocked out the ligase gene and found that transformation was reduced about six-fold. However an undergraduate student in our lab spent last year trying unsuccessfully to see evidence of the ligase activity, and she could not replicate this six-fold reduction.
This ligase needs ATP as a source of energy for its ligation reaction. But as far as we can find out, there is no ATP in the periplasm. In fact, the periplasm contains phosphatases that would cut the 'P' off of any ATP that found its way into the periplasm. One solution would be to have the ligase arrive in the periplasm already loaded with ATP. This is consistent with how such enzymes act - they first form a covalent bond with ATP, and then look for DNA to act on. But I don't know if the machinery that transports enzymes into the periplasm could use a ligase that had already assembled with its ATP. Furthermore, such an 'enzyme' could only act once, and it's hard to imagine that taking up DNA is so important that each molecule is worth sacrificing a whole ligase for.
Bottom line: we still have no idea what role this ligase might play in DNA uptake. If the ligase was essential for DNA uptake, explaining what it accomplishes might be seen as a test of any proposed mechanism of DNA uptake. It's easier to think of roles in the cytoplasm, but all the evidence points to action in the periplasm.
Earth Day 2014
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