Searching for new targets for natural transformation of E. coli

Now it is clear that my transformation does not use any competence homologue components. Others have reported another transformation mechanism (in which components of type IV secretion system are required) related to conjugation. We tried to search for these type IV secretion system homologue in E. coli K-12, but nothing was found. To our knowledge, only three form of HGT events: transformation, conjugation and transduction. The candidates for the former two forms of HGT can be excluded. The only linkage that we can refer to is transduction. First of all, we need to find porins which are conserved and required for double strand DNA phage binding or passing through the outer membrane. Then we will test their effect on our cryptic transformation.

Two candidates were screened out——tsx gene and ompA.

The reasons are as following
tsx:
1. involved with the permeation of ribo- and deoxy-nucleosides across the outer membrane of E. coli.
2. serves as a receptor for bacteriophages and colicins.
3. part of its protein has nucleotide affinity.

ompA:
1. non-specific diffusion channel
2. phage receptor
3. mediator of F-factor dependent conjugation (we have the phenotype indicating that cell density is important for transformation and this implies that cell-cell contact might be involved in transformation)
4. rpoS regulated. (rpoS is the only gene which has been identified to be required)

hofQ and gspD are not involved in

My result about gspD mutation has been out. I compared the gspD mutant, hofQ mutant and gspD-hofQ double mutant with the wild type. All can be transformed. I also tested hofQ mutant with Finkel's protocol and the result shows that it is true that hofQ mutant can not use DNA as a nutrient and the wild type can. Then how does DNA enter into cell in my protocol? People here questioned whether the transformation is a genetic event or not. But it is really controlled by rpoS. Another question is why cells do not use hofQ channel to transform but choose another way? It is still quite possible that we do not know the exactly transformation pathway which is under the control of rpoS although the possibility of indirect/mutual effect of rpoS on transformtaion can not be excluded. The main problem is where should I address in the next step since no candidates is available. I am lost again.

new mutant construction

Our institution has received a K-12 mutant library recently and I have assayed gspD mutant. It is OK. But I still will transfer this mutant to my strain to ensure the result with my new method, which is quicker than using P1 and even safer for getting mutant than using P1. I do not like using P1 transduction since I made no success at all with this method. So with this mutant library, I should be able to select more candidates even if gspD does not work this time again. I am trying to repeat Finkel's experiment about using DNA as a nutrient to see whether hofQ actually work in 'eating DNA' in my strain. My recent data support DNA can be used as a nutrient in ZK126. But my strain did not grow in the minimum culture even supplemented with glucose. This might be resulted of lacking of some amino acides. After adding these amino acides, I will see whether hofQ is really necessary for eating DNA.

Game over and play again

Ok, I should forget about the traditional pathway and try to find other ways. We should return back to the initial question --how does DNA pass throught the first membrane? There are two pilQ homologue in E. coli, hofQ and gspD. HofQ has been reported to be required for DNA as a nutrient, however, it is not the channel for our transformation. I will test the second candidate gspD and the primers should be on the way. If it is true, this might tell us the story of the criptic regulon gsp. If not, we will be blind again.

I discussed with my mentor about exploring real chromosomal transformation. He warned me that innumerous failures were on this direction for years before. We do not know whether sxy gene, which is required for natural transformation in H. influenzae and V. cholerae, express in E. coli and if so under which condition it can be turned on. In addition, in V. cholerae, except for sxy, HapR is required for the V. cholerae transformation. We do not know whether similar or additional regulators are required for turnning on competent genes in E. coli. We have evidences showing that rpoS is required for our transformation. HapR is under the regulation of rpoS in V. cholerae. However, hapR homologue was not found in E. coli genome.

who is the winner in sex evolution? ssDNA or dsDNA?

In prokaryotic cells, ssDNA translocation has widely been adopted in natural transformation and it seems that this type of transformation take an advantage in the evolution of prokaryotic kingdom. In contrast, oocytes receive dsDNA from germ cells and keep it as the double strand form before recombination in the eukaryotic realm. From the perspective of the whole biological evolution, it seems that DNA enter cells with dsDAN form is preferred. The question is, under natural conditions, why prokaryotic cell prefers utilizing ssDNA and degrading the other strand before recombination to making the use of both strands to keep the fidelity of recombination. If we support the idea that eukaryotic cells are evoluted from prokaryotic cells, the prokaryotic transformation should be a inferior form of sex left by the evolution process and there should be some reminiscence of transition this inferior sex behavior to the more advanced one. Considering the importance of sex in proliferation of eukaryotic species, trying to find the reminiscence would reveal some fundamental mechanisms of sex in eukaryotic kingdom. The first step to find the reminiscence is trying to confirm that dsDNA is able to enter into prokaryotic cells and keep its sexual biological activity under natural condition. Then we need to prove that the uptake dsDNA is able to recombinate with the genome DNA of the recipient cell. We know that millions of sperm cells (we can consider these are the donor DNA) are required for a successful impregnation event. If it is true that dsDNA recombination in prokaryotic cells is resemble to impregnation, the chance of successful reombination between dsDNA and genome might also not be very high. However, these accidents pioneered a new biology realm which comprises predominant species in the earth nowadays.

(Note: although no direct evidence is available for proving the dsDNA translocation, some evidences strongly support the existance of dsDNA translocation and the competition between dsDNA transfomration and ssDNA transformation under natural or non-physiological conditions. However, it lacks evidences to prove recombination between dsDNA and chromosomal DNA events under natural conditions)

The implication of non-classical transformation and how to address

I read a recently published paper about genome transplantation. In their report, genome transformation is applicable although with low frequency. They reported that only circular genome can be successfully transformed. But the mechanism of this type of transplantation is completely unkown. I was surprised to found that Dr. Smith is within the authors because I did not find his publications after twentith century. He has done perfect work in transformation of H. influenzae and his publications are thoughtful and insightful.



One teacher in our lab remind me one thing: what is artificial transformation? If there is no way to let DNA enter cell, there is no way for DNA entering. If there is a window there, the outer stress might make the window become a door. My question is that whether the mechanism of plasmid transformation is just the the window of so called 'artificial transformation'. People make this widow become bigger and then the transformation efficiency enhanced so the window was reformed to the door. Natural plasmid transformation has been widely found (here I mean the plasmid without DUS, if the plasmid contain DUS the transformation form changed to chromosomal transformation). Both H. influenzae and N. gonorrhoeae have such kind of transformation. Although it is not very clear whether this type of transformation exists in B. subtilis, the existance of recA-independent plasmid transformation implied that this type of transformation might also apply to gram positive bacteria. However, it is difficult to study this type of transformation at this moment because we do not know whether genes are involved in the regulation (since all competence genes are not required and we might not be able to use genetic methods). In addition, the low transformation frequency make it difficult to observe single competent cells.

Nevertheless, one can completely change the plasmid transformation to chromosomal transformation if the DUS sequence is found. The question is whether DUS sequence really exists in E. coli. Although there are reports which shows negative evidences from bioinformatics, still it is hard to say whether DUS sequence is really abscent in E. coli genome or whether it will appear in another form. In H. pylori, it is not known that whether special DNA uptake sequence is required for natural transformation and the mechanism of transformation in H. pylori is quite novel. We do not know whether the chromosomal transformation of E. coli make use of another novel transformation mechanism.

The following work seems to be a big project. But we need to design several simple experiments to reveal some important aspects and impact firstly. I will discuss with my mentor in detail about what and how I should address to next week.

Come back

I enjoyed a good weekend. Together with my friends, we went to Nice. It is quite a beautiful city and the sean is charming there. After that, we went to see the lavande and return back late in the night. It is quite interesting but I am a little tired now. I need to restart my work again and choose a point to dig out the mystical veil.