Coming back from holidays, my mind is still a bit slow in terms of thinking in perspective about my thesis project. Last year, worked a lot in extracting sheared genomic DNA taken up by Haemophilus influenzae. As this part of the project is over, I have to start thinking in the DNA uptake process in Acinetobacter baylyi. This is not an easy process, maybe because my mind is still recovering from holidays or because I read most of papers about this topic several months ago.
In order to put my ideas in order lets start with objectives for the following weeks-months:
- Design and generate synthetic fragments with a degenerate region and illumina adaptors.
- incubate this synthetic fragments with Acinetobacter baylyi competent cells and extract periplasmic DNA (This DNA will be sequenced).
Objective 1 is under way. I have already designed the synthetic oligos which will help me generate the fragments and a former post-doc of the lab is helping check that my design is correct.
The second objective is a bit more complicated since before working directly in this, I need to work on some preliminary objectives.
- Test natural transformation protocol of Acinetobacter baylyi
- Test self-specificity of Acinetobacter baylyi
- Test if the organic periplasmic extraction protocol works for Acinetobacter baylyi
Each of these objectives deserve a separate blog post, however, I will start with the first one.
- Test natural transformation protocol of Acinetobacter baylyi
When I was working with Haemophilus influenzae I did not had to worry about getting a strain or about which markers I use to test natural transformation. Our lab had an extensive collection of strains with different markers to use. However, with Acinetobacter baylyi that is not the case. What do I have? well…
Strains I have, include:
BD413 wild type
BD413 or ADP1 is a strain with high levels of natural transformation. It was derived from the soil isolate B4, and it has a small capsule which facilitate growing the bacteria in liquid media for easier centrifugation (Juni and Janik 1969).
ADP239 comA::nptII is a mutant strain which has a nptII cassette (KmR) which insertionally inactivates comA gene (which is the Rec2/ComEC homolog in A. baylyi) (Friedrich et al. 2001). All ADP239 strains came from BD413, which have a mutation in the pobA gene. This mutation prevents bacteria the growth in mineral media when p-hidroxybenzoate is the only carbon source (Porstendörfer et al. 1997).
ADP239 comP::nptII is a mutant strain which has a nptII cassette (KmR) which insertionally inactivates comP gene (Porstendörfer et al. 1997). ComP protein has similarities to the type IV type pilins and it has found to be essential for DNA binding as well as for natural transformation in A. baylyi (Porstendörfer et al. 1997). This mutant will be used in my project as a DNA uptake negative control.
These first three strains were kindly donated by Dr. Beate Averhoff.
Strains SDS-5 and SDS-6 were obtained through the teaching microbiology laboratory. These strains are naturally transformable since each year they were naturally transformed by undergraduate students as part as teaching classes. SDS-5 is the wildtype strain, while SDS-6 is an auxotrophic mutant of SDS-5 that is not able to grow in the absence of tryptophan.
Which is the background of strains SDS-5 and 6, well I do not know. I still need to talk to the person that used to be in charge of the microbiology labs. However, several papers have used tryptophan auxotrophs for natural transformation (e.g. Juni 1972, 1974, Rokhbakhsh-Zamin et al. 2011). Based these papers mentioned early, SDS-5 and 6 could have derived from strains BD413 (Juni 1972, 1974) or B4 (Rokhbakhsh-Zamin et al. 2011).
Problems or Complications
Natural transformation procedure in A. baylyi is apparently simple. The strain is cultured at 30 degrees overnight in LB. Then the culture is diluted 1 in 25ml in fresh mineral media and it is incubated for 2 hours more (Palmen et al 1993). Finally, the culture is incubated with DNA and it is plated in LB plates or minimal media agar with and without the appropriate antibiotic or carbon source (for minimal media).
Problems arise when mineral and minimal media are prepared. First, there is not a clear distinction between both media. I some papers, such as Palmen et al 1993 it seems that they are the same thing. Additionally, there are several variations of the media preparation and some are a long and complicated mixture of three solutions: a basal media, a trace element solution and a vitamin solution (i.e. Tschech and Pfenning 1984, Porstendörfer et al. 1997).
The first thing that I did to start working in A. baylyi was streaking strains SDS-5 and SDS-6 in a minimal media that I made following the recipe of Palmer et al. 1993, which consisted in 60 mM lactic acid, 11 mM-KH2PO4, 95 mM-Na2HPO4, 0.81 mM-Mg SO4, 37 mM NH4Cl, 0.068 mM-CaCl2, and 1.8 uM-FeSO4. Since SDS-6 grows only in the presence of tryptophan, I expected that this strain would grow in LB agar, but not in the lactic minimal media agar; while SDS-5 strain should grow in both agar media. To my surprise, that was not the case, since both strains grew normally in both agar plates.
This result could be caused by three factors:
- There is contamination and the colonies on plates are not Acinetobacter
- There are traces of tryptophan in one of the elements of the media
- I froze the strains incorrectly
To discriminate between the three, I will:
- Look at strains at microscope to see if there is contamination
- Get the auxotroph strain SDS-6 again from the teaching lab, and streak it in LB and lactic minimal media agar.
- Streak SDS-5 and SDS-6 in LB, lactic minimal media and in minimal media prepared with the recipe from the teaching lab (which is different from the recipe I used).
Update (Jan 07 2016)
- Colonies look normal, no contamination was observed
- I got another the Davis lab (they got the strain from the teaching lab) and the new SDS-6 grew on LB but not in the lactic minimal media (prepared by me). On the contrary, the SDS-6 that I got the first time grew on both LB and lactic minimal media.
- I also grew both strains in a minimal media plate prepared using the recipe from the teaching lab. Both SDS-6 and a SDS-5 grew poorly. Maybe more incubation time is needed
This results, on one hand, confirm that there is nothing wrong with the recipe I used to prepare the minimal media (which I got from Juni 1974, and Palmen et al 1993). On the other hand, they confirm that the first strain SDS-6 that I got was probably mistaken by SDS-5.
A second problem that I must solve relates to figure out which marker I could use to test natural transformation. Three markers could be used:
- DNA from strains with nptII gene (KmR) incubated with Kanamycin sensitive strains
- DNA from wild type strain BD413 (pobA gene) incubated with ADP239 strains which are unable to grow in minimal media with p-hidroxybenzoate.
- DNA from SDS-5 or BD413 incubated with SDS-6 auxotroph mutant
Problems with this markers:
- I could extract DNA from ADP239 comA::nptII or ADP239 comP::nptII and transform competent BD413 strain. However, this marker cannot be used to confirm the phenotype of the ADP239 comA::nptII or ADP239 comP::nptII strains since both have the nptII gene.
- Using pobA gene as a marker would be ideal, since this marker is the one used for natural transformation experiment done by the Averhoff group. However, I do not have the wild type ADP239 strain AAAGGRRR!!!!
- I could use SDS-5 DNA and select for growth in tryptophan but first, I must figure out what went wrong with the media, and second, knockout mutants of comP and comA are able to grow in tryptophan.
- It seems that the simplest solution would be to making my own ADP239 wild type. I could PCR the mutated pobA gene from ADP239 comA::nptII and transform BD413. The problem is that I will have to screen hundred of colonies in LB and minimal media with p-hidroxybenzoate
- I could extract DNA from ADP239 comA::nptII and ADP239 comP::nptII and transform SDS-6 auxotroph competent cells and select for KmR
mmm, it seems that the easiest thing to do would be the second option.