{"id":570,"date":"2014-12-15T14:02:00","date_gmt":"2014-12-15T14:02:00","guid":{"rendered":"http:\/\/suigenerisbrewing.com\/index.php\/2014\/12\/15\/brett-trois-a-riddle-wrapped-in-a-mystery-inside-an-enigma\/"},"modified":"2017-09-15T18:15:51","modified_gmt":"2017-09-15T18:15:51","slug":"brett-trois-a-riddle-wrapped-in-a-mystery-inside-an-enigma","status":"publish","type":"post","link":"https:\/\/suigenerisbrewing.com\/index.php\/2014\/12\/15\/brett-trois-a-riddle-wrapped-in-a-mystery-inside-an-enigma\/","title":{"rendered":"Brett Trois – A riddle, wrapped in a mystery, inside an enigma."},"content":{"rendered":"

So recently a youtube viewer of mine (Dan Aba, whose youtube channel<\/a> you need to check out if you haven’t already) turned me onto a Facebook group dedicated to sour beers. Lo and behold, the first thread I see on the forum is one claiming that Brettanomyces trois<\/i>\u00a0is actually a Saccharomyces<\/i> yeast! This info didn’t just come from nowhere;\u00a0Lance Shaner of Omega Yeast Labs<\/a>\u00a0sent of the strain to a friend for sequencing, and the sequence came back Saccharomyces<\/i>. Unfortunately, the sequence quality was poor and the sample appears to be a mix of two strains – so I thought it was time to do my own investigation.<\/p>\n

The process I followed was fairly straight forward:<\/p>\n

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  1. I grew up B. trois<\/i>\u00a0from my yeast bank, overnight in a 37o<\/sup>C shaking culture – 1.040 wort + penicillin and streptomycin (to ensure a bacteria-free culture, not because my stocks are dirty)<\/li>\n
  2. I took some images to assess morphology of the yeast<\/li>\n
  3. I isolated some DNA and sequenced the Internal Transcribed Spacer (ITS) and part of the small (18s) rRNA gene to identify the yeast, using an optimized version of what I was doing in my previous posts.<\/li>\n<\/ol>\n
    So what did these experiments show? . . . . . . . Answer (and details) below the fold.
    \n    <\/a><\/p>\n
    \n

    And the species is…<\/h3>\n

    So normally I would leave the big reveal to the end, but for those who want to avoid the nitty-gritty the answer is…<\/p>\n

    Brettanomyces trois<\/i>\u00a0– at least the version sold by White Labs (WLP644) – is a Saccharomyces<\/i>.<\/b> It is most likely Saccharomyces cerevisiae<\/i>, but the genetic information doesn’t nail that down completely.
    \n
    \n<\/b>So, how do I know?<\/p>\n

    \n

    Morphology<\/h3>\n<\/div>\n
    In my previous posts I have mentioned that morphology is not overly useful for identifying yeast<\/a> – sometimes it can shorten the list of possibles, but on days like today it can also act to confuse things further. Keep in mind that this culture started off as a single colony; so its unlikely to be a blend of two or more yeasts. Even so, I see both sacc<\/i>-like and brett<\/i>-like morphologies.\u00a0Saccharomyces<\/i>\u00a0cells tend to be slightly elliptical and symmetrical across the short and long axes. When they divide, Saccharomyces<\/i> usually buds just to the side of one pole of the cell – i.e. they look exactly like the cells on the right side of the below montage. In contrast, Brettanomyces<\/i>\u00a0tends to show a range of cell morphologies – everything from Saccharomyces<\/i>-like ovoids through to chains of elongated cells. Moreover, Brettanomyces<\/i>\u00a0is a little less rigorous about where it forms buds – just like the cells on the left side of the below montage…<\/div>\n
    <\/div>\n\n\n\n\n
    <\/a><\/td>\n<\/tr>\n
    Four WLP644 – in budding (top) and stationary (bottom) phase<\/b>
    \nLeft: yeast with brett<\/i>-like morphology
    \nRight: yeast with sacc<\/i>-like\u00a0morphology<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n
    \n

    For those who want to get a better idea of the diversity of cell shapes and sizes, here is a field of view that does a good job highlighting the various morphologies that I observed in the culture of WLP644.<\/p>\n<\/div>\n\n\n\n\n
    <\/a><\/td>\n<\/tr>\n
    Click for Full-Size<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n
    <\/div>\n
    Clearly microscopy isn’t going to solve this riddle, so onto the DNA.<\/div>\n
    \n

    Ribosomal Sequencing<\/h3>\n

    The next step was to sequence two parts of the yeast’s ribosome in order to positively identify the genus and species of this yeast. I have blogged about these methods extensively (posts 1<\/a>, 2<\/a>, 3<\/a> and 4<\/a>), so I’m not going to go into all of the details, but long story short I sequenced two parts (ITS and small ribosomal RNA) of the yeast genome useful for identifying different species of yeast; unfortunately, the small ribosomal sequence did not work, so all I have is the ITS<\/p>\n

    \n

    The ITS sequence is:<\/p>\n<\/div>\n\n\n\n\n
    Internal Transcribed Space (ITS1)<\/b><\/td>\n<\/tr>\n
    >WLP644 ITS Sequence<\/span>
    \nCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGCAGAATTCCGTGAAT<\/span>
    \nCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCAGGGGGCATGCCTGTT<\/span>
    \nTGAGCGTCATTTCCTTCTCAAACATTCTGTTTGGTAGTGAGTGATACTCTTTGGAG<\/span>
    \nTTAACTTGAAATTGCTGGCCTTTTCATTGGATGTTTTTTTTTCCAAAGAGAGGTTT<\/span>
    \nCTCTGCGTGCTTGAGGTATAATGCAAGTACGGTCGTTTTAGGTTTTACCAACTGCG<\/span>
    \nGCTAATCTTTTTTTATACTGAGCGTATTGGAACGTTATCGATAAGAAGAGAGCGTC<\/span>
    \nTAGGCGAACAATGTTCTTAAAGTTTGACCTCAAATCAGGTAGGAGTACCCGCTGAA<\/span>
    \nCTTAAGCATATCAATAAGC <\/span><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

     <\/p>\n

    The next step is obviously to identify to which species WLP644 is the most closely related to. I’ve done this in two ways – my old method of performing a BLAST search<\/a>\u00a0with settings that will hopefully pull quality matches, but as my older posts show, this method doesn’t work well due to the high abundance of other yeast sequences – notably Picha<\/i>\u00a0strains – which tend to bias the results. None-the-less, based on the BLAST search, WLP644 is:<\/div>\n
    \n