Eureka, I begin the yeast basic post series with today’s post which is all about the family tree of Brettanomyces. The main message here is how the different Brettanomyces strains are connected to each other and which strains are the same. Be aware, there is a lot of science in this post. Lets begin with a question.
How would you determine the relationship between people? Easy question but the way to do it is not that simple. Of course you could ask some of the people about their relationship to each others and then construct a family tree. You might fail with this approach if you have like: Who is the father of this child? What if you get two fathers? How would you construct a family tree then? In such cases, genetics helps. One way to solve this problem would be to sequence the childs and the two fathers DNA and compare them. One would expect less differences between the DNA of the child and father because the child inherited part of the fathers DNA. This approach can be used to construct relationships and build a family tree (phylogenetic tree). In this case you take the same DNA parts of different people, in our case here Brettanomyces, and compare them.
However, there is a major difference. The family tree of Brettanomyces is very old with a lot of generations. Imagine that your parents lived a million years ago and raised an ancestor of you back then. This might sound silly to you because they raised an ancestor of you and are still your parents. Normally, you would call your direct ancestors your parents, right? However, in this case you are a clone of your ancestor with nearly the exact same DNA. Meaning, the DNA of all your ancestors back to your parents are the same as yours today. If you would compare all the DNA of all your ancestors back to your parents, you might find only very minor differences. Only your parents will have different DNA. And this makes it possible again to create a phylogenetic tree.
I like to emphasize that there could be differences in the DNA between the ancestors for sure. A lot of mutations happen every day in a cell. Can’t remember the exact number but its huge… Anyway, there is the probability that some of the DNA differ once in a while. Some changes might work, some are lethal for a cell (they mess up the functionality of a cell). This already shows that only some mutations are inherited (lethal mutations can’t be inherited). To get back to the phylogenetic tree. The problem now is to choose the correct piece of DNA. If you chose a part of DNA with a lot of mutations, you would not be able to construct a phylogenetic tree because the changes in the DNA due to inheritance is interfered with random mutations. This leaves regions where only a few mutations happen during time. Additionally, if you chose a region were not only few mutations happen but some of them get lost due to lethality, you have yourself regions were only very few mutations happen at the end. I will not go into further detail now. At the end you chose regions in the 26S ribosomal subunit or regions between different rRNA, called internal transcribed regions (ITS).
The ribosome is the machinery that produces proteins in a cell and is therefore very important. If the ribosome does not work (due to mutations for example) it can’t synthesize proteins and the cell might die. A ribosome consists of several rRNA (ribosomal RNAs). Imagine the different rRNA to be kinds of Lego bricks forming a functional ribosome at the end. One of them is the 26S rRNA. Just a clarification for all the people out there wondering about the 26S and not 28S (typical for eukaryotes), the 26S is the homolog of the 28S in yeast, plants and protozoans.
Other rRNAs necessary for the ribosome are the 18S, 5.8 S and many others. At the genetic level, the 18S rRNA and 5.8S rRNA are separated by a region called ITS1 (internal transcribed region). The 5.8R rRNA and the 26S rRNA are separated by a region called ITS2. This means that the genes for these rRNAs are located on the DNA level as following:
18S – ITS1 – 5.8S – ITS2 – 26S
At the end, you chose the ITS1 and ITS2 or the 26S gene for the phylogenetic tree. Enough of theoretical background here. Lets get into the practical part.
Phylogenetic tree of Brettanomyces based on ITS1/ITS2 comparison:
All the sequences can be found on any genebank of your choice. I used the NCBI database to get the different sequences from six different Brettanomyces strains:
B. custersianus, B. naardenensis, B. clausenii, B. anomalus, B. bruxellensis and B. lambicus
After you got yourself the sequences, you compare them. This is called alignment in bioinformatical terms. How an alignment looks like is shown in Fig 1.
What you see there are the different sequences from the different Brettanomyces species at the top with the labels on the left site. Each of the four bases of the DNA (A,T, G, C) has its own color to make it easier for the human eye to compare. Lets make an example. Lets have a look at the very left column. The sequence of B. naardenesis starts with an A, all the other species have a G at this position. If all the bases are the same, like in the next column, you get a 100% conservation at the bottom. Meaning, that all the bases in the different species have the same base at this position. In our example, there is one A and 5 G’s. The conservation here is not 100%. Now, the interesting parts here are the regions with a lower conservation. These are the regions were changes happened during the time and where you can gain information about the relationship.
The next step is to create a phylogenetic tree from this alignment. I have no idea how the program does it exactly. I assume the program looks for patterns how the different conservation states occurred. What you get at the end is the following picture (Fig 2).
What you can see here is that there are four branches. One for B. custersianus, one from B. naardenensis, one shared by B. clausenii and B. anomalus and one shared by B. bruxellensis and B. lambicus. Each branch represent a family, called species in biology. Basically, there are four different Brettanomyces species. There is another one, B. nanus, but I could not find any ITS1/2 data for this particular strain. Another thing here is that B. bruxellensis, B. lambicus and B. clausenii and B. anomalus are from the same species. B. bruxellensis and B. lambicus, B. clausenii and B. anomalus are synonyms for the same species. I had a look at the alignments of these two strain pairs and they were exactly the same (100% conservation). Genetically, the B. lambicus/B. bruxellensis and B. clausenii/B. anomalus ITS regions are the same. All of you out there working with Brettanomyces might know that the flavor profile of B. lambicus and B. bruxellensis are not the same. Looking only at the flavor profile you might categorize the two strains in different species. However, this is not the scientific way to categorize strains into species. Its done on a genetic level like shown here.
Phylogenetic tree of Brettanomyces based on 26S rRNA comparison:
The process is basically the same as mentioned above, this time with the 26S rRNA regions.
Now with the B. nanus, you can see now that there are five different Brettanomyces species (Fig 2).
These trees are not new and already published in different publications. The results from these trees basically led to the definition of the five accepted Brettanomyces species known today:
B. bruxellensis (B. lambicus as synonym), B. anomalus (B. clausenii as synonym), B. custersianus, B. nanus and B. naardenensis.
To summarize, there are five different Brettanomyces species accepted today. Of course there could be new species to be discovered. To be recognized as a new species it has to form a new branch in the phylogenetic tree, not sharing an already existing one.
I was quite fascinated how easy it is to get yourself a phylogenetic tree of Brettanomyces within a reasonable amount of time. Of course, there are publications about this already. However, it is much more exciting to be able to replicate the results yourself. And a very good exercise. I hope it is somewhat clear what happened here and how information of DNA sequences can be used as well.
Next posts to come: general overview about Brettanomyces, further information about the individual Brettanomyces strains and another genetic post about the comparison of a Brettanomyces against a Saccharomyces genome. Stay tuned!