Identification of new species - (Nov/22/2012 )
I isolated bacteria from a dog, sequeneced the 16S rRNA gene and aligned it (similarity after manual correction and BLAST search ~96%). Then, I sequenced two other protein coding genes yielding a homology of <90% to the closest relative. I did the whole procedure twice. A few month ago, I isolated bacteria, selected one single colony and sequenced it. Now, I isolated again and selected three colonies. I streaked them on plate several times and again sequenced the genes.
I am wondering why all the sequences from all the colonies are 100% identical. Shouldn't there be slight differences, i.e. different strains?
Will people believe that I isolated twices if the sequences are fully identical?
Thanks a lot!
Perhaps you used highly conserved regions of the DNA within that species.
Why not? I have a fungal isolate from Australia which has 100% identity with another from Japan and other from China, and based on the ITS which is much more variable.
Did you isolate the second time from dog too? Same medium?
Some species have a plethora of closely related strains or within the same species, almost like a gradient of similarity based on 16S. Others however, may have the exact same 16 S or maybe just one bp different and being different species. The last case is not very common but there are some cases that need extra sequencing of some other genes to confirm.
yes, I did everything the same way.
16S was completely the same for all isolates. Then, I sequenced gyrA and rpoB but the sequences were again 100% identical. Okay, if your samples from Australia and China did not show any differences for ITS, this might also be the case for my isolates.
I will conduct ERIC fingerprinting, maybe yielding bands of different length. What do you think of this approach?
Another question concerning the identification of new species:
I read a lot of papers and the authors only sequenced the 16S rRNA gene. In addition, they conducted a lot of biochemical tests and also cell wall murein analysis and lipid analysis. Are the latter two essential when describing a new species from Corynebacterium genus?
The method papers mentioned in the publications are very old. For example, they use paper chromatography and to be honest, it will be very hard for me to reproduce the methods since I have never done this before.
Can I replace these analyses by sequence data of 4 genes instead of only one (16S rRNA)? I will add results from API Coryne but I have no idea about cell wall and lipid analysis.
If you want to describe a new species, it must be "validly published," which means you need to describe it, including all of the information (in a genus specific way) needed by that group of organisms. In the past, this often required many tests that are awkward to perform, including things like TEM images and serology. You'll have to check recent articles in the canonical journal for this, which is the International Journal of Systematic and Evolutionary Microbiology (IJSEM). Things published there typically get good reviewing and are automatically "validly published." Consulting with the editor would be a good first step.
Check http://www.bacterio.cict.fr/ for the newest described species of the genus and see what they did. Requirements may be variable depending on the taxa. There are several Corynebacterium described as new in the last decade