Example phylogenetic analysis : strain ES-2, a.k.a. Eubacterium thermomarinus

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In about 1990, an organism (designated ES-2) was isolated from a deep-sea hydrothermal vent sample. ES-2 grows heterotrophically at 65C. A lipid analysis of the isolate was unusual, containing a number of apparently branched lipids as well as fatty acids. Electron microscopy and standard microbiological tests were not helpful in identifying the organism.

Phylogenetic analysis of the organism was performed essentially as described in class. DNA was isolated from cells and the ssu-rRNA was amplified by PCR using 'universal' primers near both the 5' and 3' ends of the gene. The amplified DNA was cloned and sequenced. Here is the sequence, in GenBank format:

       LOCUS       Eub.tmarin     893 bp     RNA               12-FEB-1993
       DEFINITION  Eubacterium thermomarinus.
       ACCESSION  diversity:1834:811629716
       AUTHORS  Pledger,R.J., Brown,J.W., Hedrick,D.R., Pace,N.R., White,D.C.
       REFERENCE   1  TITLE Preliminary characterization of a rod-shaped thermophilic, 
          auth line 2:and Baross,J.A.
          title line 2:anaerobic eubacterium isolated from a submarine
          title line 3:hydrothermal vent environment
       BASE COUNT     345 a    335 c    465 g    263 t   1438 others
       ORIGIN 
          1  GCCUAACACA UGCAAGUCGA GCGGGUAGGU GCUUUAAUGA ACCUUCGGGG
         51  GAUUUAAAGU ACUGAAAGCG GCGGACGGGU GAGUAACGCG UGGGGAACCU
        101  GCCCUAUGCA GGGGGAUAGC CUCGGGAAAC CGGGAUUAAU GGCCCAUAAC
        151  ACUUAUGGAC CGCAUGGUGC AUAAGUCAAA GCGUUUAGCG GCAUAGGAUG
        201  GCCCCGCGUC CCAUUAGCUA GUUGGUGAGG UAACGGCUCA CCAAGGCGAC
        251  GAUGGGUAGC CGGCCUGAGA GGGUGGACGG CCACACUGGG CCUGAGACAC
        301  GGCCCAGACU CCUACGGGAG GCAGCAGUGG GGAAUAUUGC ACAAUGGGCG
        351  AAGCCUGAUG CAGCGACGCC GCGUGAGCGA AGAAGGCCUU CGGGUCGUAA
        401  AGCUCUGUCC UAAGGGAAGA AAGAUGUCGG UACCUUAGGA GGAAGCCCCG
        451  GCUAACUACG UGCCAGCAGC CGCGGUAAUA CGUAGGGGGC GAGCGUUGUC
        501  CGGAAUCACU GGGCGUAAAG GGAGCGUAGG CGGCCCUGCA AGUCAGGUGU
        551  GAAAGGCAUC GGCUCAACCG AUGUGAGCAC UUGAAACUGU AGGGCUUGAG
        601  UGCAGGAGAG GAGAGCGGAA UUCCUAGUGU AGCGGUGAAA UGCGUAGAUA
        651  UUAGGAGGAA CACCAGUGGC GAAGGCGGCU CUCUGGACUG UAACUGACGC
        701  UGAGGCACGA AAGCGUGGGG AGCGAACAGG AUUAGAUACC CUGGUAGUCC
        751  ACGCCGUAAA CGAUGAGUGC UAGGUGUUGG GGGGUAACUC CUUCAGUGCC
        801  GCAGUUAACA CAUUAAGCAC UCCGCCUGGG GAGUACGGUC GCAAGACUGA
        851  AACUCAAAGG AAUUGACGGG GACCCGCACA AGCAGCGGAG CAUGUGGUUU
        901  AAUUCGAAGC AACGCGAAGA ACCUUACCAA GACUUGACAU CCUCCGGACG
        951  GCUCUGGAGA CAGGGCUUUC CCUUCGGGUA CUGGAGAGGC AGGUGGUGCA
       1001  UGGUUGUCGU CAGCUCGUGU CGUGAGAUGU UGGGUUAAGU CCCGCAACGA
       1051  GCGCAACCCU UGCCUUUAGU UGCCAUCAGG UAAAGCUGGG CACUCUAUGA
       1101  GGACUGCCGG CUAAAAGUCG GAGGAAGGUG GGGAUGACGU CAAAUCAUCA
       1151  UGCCCCUUAU GUCUUGGGCU ACACACGUGC UACAAUGGCC GGUACAGAGG
       1201  GCAGCGAACC CGUAAGGGGG AGCGAAUCCC AAAAAGCCGG UCCCAGUUCG
       1251  GAUUGUGGGC UGCAACUCGC CCACAUGAAG CUGGAGUUGC UAGUAAUCGC
       1301  GAAUCAGAAU GUCGCGGUGA UGCGUUCCCG GGUCUUGUAC ACACCGCCCG
       1351  UCACUCCAUG AGAGUCGGCA ACACCCGAAG CCAGUGGGCU AACCGAAGGA
       1401  GGCAGCUG
       //     

And here is the secondary structure of the RNA:

ES2 RNA
ES-2 ssu-rRNA, Jim Brown

With the sequence in hand, the next step in the process is to align the sequence with the ssu-rRNA database - this was originally done by hand in a local database, but these days can be done automatically at the Ribosomal RNA Database. After this, the next step is to calculate phylogenetic trees. First, a "representative" tree was generated to see which major phylogenetic group of the Bacteria this organisms seems to belog to:

broadTree
Phylum-scale tree including ES-2, Jim Brown (generating using the RDP II)

The sequence seems most closely related to the representative of the phylum Firmicutes (represented by Bacillus mycoides). So, the next step is to generate another tree using representative sequences from the Firmicutes. At the time this analysis was done, only a few such sequences were available - now there are thousands low G+C Gram-positive sequences in the databank!

midscale tree
Tree of the Firmicutes, including ES-2, Jim Brown (Generated using the RDP II)

The tree shows that ES-2 is a member of the Clostridium/Eubacterium group of the Firmicutes. So a final tree with representatives of this group, and some especially close relatives identified using BLAST was generated:

finescale tree
Fine-scale phylogenetic tree of ES-2, Jim Brown (generated using the RDP II)

Interpretation

ES-2 is a member of the Clostridium/Eubacterium group of the Firmicutes, and particularly close (probably deserves to be in the same genus or even species) to Caloranaerobacter azonensis, a more recent deep sea vent isolate. At the time of this anaylsis (but no longer), organisms from this group that produce spores were considered to be in the genus Clostridium, those that did not were named Eubacterium. Because of the environment it was isolated in, the new species was named Eubacterium thermomarinus.

What good was this information?

This information was very useful at the time - we were interested in identifying organisms that were very distnict from those already in hand for anaylsis of RNase P RNA structure. Given that this was basically a Clostridium, from which we already had several sequences, this organism became a low priority and was not pursued further. Knowing the phylotype of ES-2 prevented us from spending a fair amount of effort for what would have been trivial gain.