2007年9月18日星期二
Come back and prepare for my future experiments
I just come back from my vocation. It was a nice journey. I have visited Paris, Berlin and Amsterdam. Now I should sit down and think what I should do next.
2007年9月3日星期一
2007年8月31日星期五
mystery is still mystery
I will go out for a while. I hope I can finish constructing the desired mutants and plasmids before my depature.
I discussed with someone who works about T4 phages. He said that he knows only the injection mechanism about phage infection. Actually, people do not know much about the mechanism about phage infection process as well. He said that the inner membrane receptor has not be found in T4 infection. In addition, porins found to be the receptor of phages does not function as a channel through which phage DNA pass through. Phages use their special ways to circumvent the outer membrane. We also talked about something about porins. He gave me two suggestions: 1. extracting the outer membrane proteins and doing the pull down experiment to see which outer membrane proteins interact with DNA 2. predict outer membrane proteins and screen the candidates by some special programs. Both ways are highly risky and chance is not high. The story about this type of transformation is still a mystery.
I discussed with someone who works about T4 phages. He said that he knows only the injection mechanism about phage infection. Actually, people do not know much about the mechanism about phage infection process as well. He said that the inner membrane receptor has not be found in T4 infection. In addition, porins found to be the receptor of phages does not function as a channel through which phage DNA pass through. Phages use their special ways to circumvent the outer membrane. We also talked about something about porins. He gave me two suggestions: 1. extracting the outer membrane proteins and doing the pull down experiment to see which outer membrane proteins interact with DNA 2. predict outer membrane proteins and screen the candidates by some special programs. Both ways are highly risky and chance is not high. The story about this type of transformation is still a mystery.
2007年8月11日星期六
To survie, everything is possible
No question has been resolved but more questions come out.
1. Is it safe to say that pilQ and comEC are the only channels for DNA passing through the outer membrane and inner membrane now?
For the inner membrane comEC/rec-2 might not be the only channel through which exogenous DNA passes through. My previous experiment clearly shows that transformation did not reduced at all in comEC/ycaI homologue mutant. For the outer membran, pilQ homologue (hofQ and gspD) are not required and so the outer membrane is not exclusive.
2. Stress induce E. coli transformation or nutrient limitation induces this transformation?
Transformation was regulated by rpoS, so transformation should be a response to the general stress. My previous experiment also implicated that crp might play similar role in transformtion. Unfortunately, I did this experiment only one time and after that the mutant was contaminated and always after transformation only white colonies appear on plates and plasmid can not be isolated from these white colonies. So I am not very sure about this result. But I did this experiment with the positive control (Wild type), negative control (rpoS mutant). I used two independent markers to mark the transformant: ampicillin resistance and red fluorescence. Even only with one time result, still I think this should be true. It seems that transformation is regulated both by stress and nutrient limitation. I can just imagine what it might be but it is hard to prove anything since I do not know throug which channels DNA enter into cells.
3. the relation among competence, nutrient limitation and stress. Difficult to distinguish and why we distinguish them?
I repeated Finkel's experiment. Although they lack some essential controls, their conclusion that hofQ is required for DNA used as nutrient is true. I added a control in this experiment, that is let cells grow in minimum culture added glucose to see whether the mutant can eat glucose.
We proposed if hofQ mutant (cloramphnicol resistance) behave the same as wild type cell, when we put the same number of wild type cells and mutant cells in the culture supplemented with glucose, the number of cells on LB plate should be 2 times of that on LB supplemented with cloramphinicol. To our surprise, we get 0 clonies on the plates with chloramphicol and more than 1000 transformants on LB plates (culture was serial diluted). That is wild type cells gain growth advantage even in minimum culture with glucose (they have reported such phenomenon in LB long ago). And such phenomenon was also found in minimum culture added DNA.
Later, I found that hofQ mutant with cat gene cassette grow slower than that of wild type cells in minimum culture with glucose and after I delete the cat gene cassette away, the mutant return back to the normal growth. The mutant without cat cassette can not 'eat' DNA either. Kolter's group has reported that 10-day-old cell can suppress the growth of 1-day-old cell and this phenomenon was regulated by rpoS more than 15 years ago. It is possible that hofQ mutant with cat cassette grow slower (just like 1-day-old cell) and its wild type competitor (like 10-day-old cell) grow quicker and take grow advantage. These results implied that competence homologue gene might be involved in both nutrient acquisition and general stess resposne.
Recent opinions to the evolution of genetic transformation incline to the theory of genetic exchange to gain genetic diversity to cope with stresses in environment, although DNA nutrient theory has also been proposed by some people. In my opinion, the two theory should not conflict, considering that nutrient limitation should be the biggest stress for bacteria in environment.
1. Is it safe to say that pilQ and comEC are the only channels for DNA passing through the outer membrane and inner membrane now?
For the inner membrane comEC/rec-2 might not be the only channel through which exogenous DNA passes through. My previous experiment clearly shows that transformation did not reduced at all in comEC/ycaI homologue mutant. For the outer membran, pilQ homologue (hofQ and gspD) are not required and so the outer membrane is not exclusive.
2. Stress induce E. coli transformation or nutrient limitation induces this transformation?
Transformation was regulated by rpoS, so transformation should be a response to the general stress. My previous experiment also implicated that crp might play similar role in transformtion. Unfortunately, I did this experiment only one time and after that the mutant was contaminated and always after transformation only white colonies appear on plates and plasmid can not be isolated from these white colonies. So I am not very sure about this result. But I did this experiment with the positive control (Wild type), negative control (rpoS mutant). I used two independent markers to mark the transformant: ampicillin resistance and red fluorescence. Even only with one time result, still I think this should be true. It seems that transformation is regulated both by stress and nutrient limitation. I can just imagine what it might be but it is hard to prove anything since I do not know throug which channels DNA enter into cells.
3. the relation among competence, nutrient limitation and stress. Difficult to distinguish and why we distinguish them?
I repeated Finkel's experiment. Although they lack some essential controls, their conclusion that hofQ is required for DNA used as nutrient is true. I added a control in this experiment, that is let cells grow in minimum culture added glucose to see whether the mutant can eat glucose.
We proposed if hofQ mutant (cloramphnicol resistance) behave the same as wild type cell, when we put the same number of wild type cells and mutant cells in the culture supplemented with glucose, the number of cells on LB plate should be 2 times of that on LB supplemented with cloramphinicol. To our surprise, we get 0 clonies on the plates with chloramphicol and more than 1000 transformants on LB plates (culture was serial diluted). That is wild type cells gain growth advantage even in minimum culture with glucose (they have reported such phenomenon in LB long ago). And such phenomenon was also found in minimum culture added DNA.
Later, I found that hofQ mutant with cat gene cassette grow slower than that of wild type cells in minimum culture with glucose and after I delete the cat gene cassette away, the mutant return back to the normal growth. The mutant without cat cassette can not 'eat' DNA either. Kolter's group has reported that 10-day-old cell can suppress the growth of 1-day-old cell and this phenomenon was regulated by rpoS more than 15 years ago. It is possible that hofQ mutant with cat cassette grow slower (just like 1-day-old cell) and its wild type competitor (like 10-day-old cell) grow quicker and take grow advantage. These results implied that competence homologue gene might be involved in both nutrient acquisition and general stess resposne.
Recent opinions to the evolution of genetic transformation incline to the theory of genetic exchange to gain genetic diversity to cope with stresses in environment, although DNA nutrient theory has also been proposed by some people. In my opinion, the two theory should not conflict, considering that nutrient limitation should be the biggest stress for bacteria in environment.
2007年8月1日星期三
plasmid transduction
This morning, I read some referrences about plasmid transduction. Plasmid transduction is using phages extracted from hosts harboring a plasmid transducts a receptor strain and screen the transductants expressing the plasmid marker. This means that the plasmid has recombined to the phage when the phage enter into the host, and that plasmid can be carried into cells on the phage vector.
It might not be surprising from the perspective of bacteriophage infection. But the addition of plasmid can change the infection behavior of phage infection and different plasmids have different abilities of plasmid transduction while the recombination frequency in the host cell keep the same. Whats more, recA is not required for plasmid transduction behavior. All these phenomenon strongly suggest that plasmid in itself affect the infection process.
For natural transformation, phages may know more than us. Maybe phage just makes full use of this mechanism to circumvent the two membrane barrier. Although phages adopt various strategies to pass through the barriers, in the final stage they will have to let their DNA/RNA to cope with the plight. Some lucky phage DNA can be sent to periplasm and face only the inner membrane while some other phage DNA has to face the first membrane as soon as they were released from the phage capsule. Nevertheless, phage DNA (I thinkt there should be no difference between plasmid DNA) must have learned the capability of passing through membranes and capsules and proteins encoded by genes on phage genome just aid DNA entering and enhance the chance of entering and surviving in the host.
Recently, someone argued that RNA virus might be the ancestor of all organism in the earth. Considering that RNA virus is not stable and can be easily degraded, I support the idea that DNA virus should be the ancestor, locating at the root of the evolution tree. It is possible that DNA gradually evolved to DNA virus.
It might not be surprising from the perspective of bacteriophage infection. But the addition of plasmid can change the infection behavior of phage infection and different plasmids have different abilities of plasmid transduction while the recombination frequency in the host cell keep the same. Whats more, recA is not required for plasmid transduction behavior. All these phenomenon strongly suggest that plasmid in itself affect the infection process.
For natural transformation, phages may know more than us. Maybe phage just makes full use of this mechanism to circumvent the two membrane barrier. Although phages adopt various strategies to pass through the barriers, in the final stage they will have to let their DNA/RNA to cope with the plight. Some lucky phage DNA can be sent to periplasm and face only the inner membrane while some other phage DNA has to face the first membrane as soon as they were released from the phage capsule. Nevertheless, phage DNA (I thinkt there should be no difference between plasmid DNA) must have learned the capability of passing through membranes and capsules and proteins encoded by genes on phage genome just aid DNA entering and enhance the chance of entering and surviving in the host.
Recently, someone argued that RNA virus might be the ancestor of all organism in the earth. Considering that RNA virus is not stable and can be easily degraded, I support the idea that DNA virus should be the ancestor, locating at the root of the evolution tree. It is possible that DNA gradually evolved to DNA virus.
2007年7月31日星期二
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)
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)
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