Determining the phylotype of the YNP pink filaments organism

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PDF Reysenbach AL, Wickham GS & Pace NR 1994 Phylogenetic analysis of the hyperthermophilic pink filament community in Octopus Spring, Yellowstone National Park. Appl. Env. Microbiol. 60:2133-2199

Question being asked : What is the phylogenetic identity (phylotype) of the pink filamentous organism?

Octopus Spring
Octopus Spring, Yellowstone National Park (JWBrown)

Described in the 1960's by Thomas Brock (the namesake of the very good General Microbiology textbook published by Prentice Hall), there is an abundance of pink filamentous growth in many neutral pH Yellowstone hot springs, including Octopus Spring. Early attempts to label these filaments by feeding them radioactive organic compounds failed, as did early attempts to extract nucleic acids, causing some to suggest that they weren't alive but just the dead remains of mesophilic groundwater organisms that had been regurgitated by the hot spring. Thermus aquaticus (which is often also pink) was isolated as a by-product of attempts to cultivate these filaments, but it turns out Thermus is a minor constituent of this microbial community.

Pink Filaments
Pink filaments in the outflow of Octopus Spring (JWBrown)

In this 1994 paper by Anna-Louise Reysenbach and Gene Wickham from Norm Pace's lab, the pink filaments were indentified without cultivation by molecular phylogenetic analysis.

Pink filament biomass was collected following growth for several weeks on square, flat cotton filter pads placed in situ, to make sure the organisms actually grew in that environment rather than being groundwater organisms brought, dead, to the surface in the hot spring. These "furnace filters" quickly became colonized by the pink filamentous growth.

Fig 1
Scanning electron microgrpah of pink filaments grown in situ in Octopus Spring (Fig 1, Reysenbach et al 1994 AEM 60:2133)

DNA was isolated from a washed pink filament sample, and ssu-rDNA was amplified by PCR. Interestingly, only universal or bacterial-specific primers yielded PCR products; archaeal-specific primers did not amplify anything. This probably is the result both of the fact that the archaeal-specific primers weren't that good, and because this environment probably really is predominated by Bacteria rather than Archaea. The PCR products were cloned into a plasmid vector before analysis; cloning is required when analyzing PCR products from natural samples because they will be a mixture of sequences; cloning is used to separate this population into individual sequences. 35 clones were obtained.

The sequences were sorted in groups of related clones using "T-tracts", and a single clone from each set of highly-similar seuences was sequenced; there's little reason to sequence a slew of identical or nearly identical sequences. This was necessary because, at the time of these experiements, sequencing was a lot more work (by orders of magnitude) than it is today. "T-tracts" are standard sequencing reactions, but instead of performing the reactions to identify all 4 bases (A,G,C, and T), only the "T" reactions are performed. These are run side-by-side on a gel, and the T-tract patterns (a sort of fingerprint) are sorted to identify sets of identical or nearly identical sequences. A representative clone from each unique type (there were only 3 types) were fully sequenced. T-tracts are not used any more, but were run in the "old days" when you had to run each of the 4 sequencing reactions separately and run them on separate lanes of a gel, and had to do several runs from different primers to get the entire PCR product sequence. In todays automated sequencing systems, all 4 reactions are run in the same tube and run on a single lane of a gel.

Most of the sequences (26 of the 35 clones obtained) were a novel sequence (named "EM17"), related to what was at the time a newly-isolated organism, Aquifex pyrophilus, a hyperthermophilic hydrogen oxidizer. There were two other sequences that showed up less often among the clones:

EM tree
Phylogenetic tree of the three EM sequences (Fig 2, Reysenbach et al 1994 AEM 60:2133)

EM19 (2/35) is a less-close relative of Aquifex, and EM3 (7/35) is a relative of Thermotoga.

Consistent with EM17 being related to Aquifex are some details of the secondary structures inferred from the ssu-rRNA. The authors show this in a "hypervariable" region of the ssu-rRNA. If you think back to ourdiscussion long ago about molecular phylogenetic clocks, this region might represent a "second hand". Not only does the sequence change very quickly over evolutionary history, but so does the secondary structure. What's clear is that the structure of this region is much like tgat of Aquifex pyrophilus, a 6bp proximal helix and a 5bp stem/loop flanking an asymetric internal loop with conserved sequence ("CUC" and "A". Notice how the sequences in the helices are quite different, but all changes are conistent with the secondary structure. The only difference in the secondary structures are the sizes of the terminal loops. In contrast, the same region in Thermotoga or Escherichia are very different in both sequence and structure.

fig 4
Secondary structure of a hyoervariable region of the ssu-rRNA of EM 17 compared to other Bacteria. (Fig 2, Reysenbach et al 1994 AEM 60:2133)

The question then was, which of these three sequences (if any) are from the pink filamentous organism? To determine this, a fluorescently-labeled oligonucleotide probe complementary to a species-specific (highly variable) region of the EM17 ssu-rRNA sequence was used to probe environmental samples for the EM17 organism; this is called fluorescent in situ hybridization, or FISH. The EM17 probe did fluorescently-label the pink filaments, and not other inhabitants of the sample, showing that the EM17 ssu-rRNA sequence really is that of the pink filament microbe. The EM19 and EM3 specific probes failed to hybridize to anything observable in the samples.

Fig 3
Fig 3 (Reysenbach et al 1994 AEM 60:2133)

So, in conclusion, the authors have determined the phylotype of the pink filaments, which turns out to be a relative of Aquifex.

A note about informal clone names

By the way, "EM" stands for Electric Monk, the haywire labor-saving device described in "Dirk Gently's Holistic Detective Agency" by Douglas Adams. When the Electric Monk is introduced in the story, he is stuck because he believed (believing is, after all, the function of an Electric Monk) that everything around him is a uniform shade of pink, making distinguishing any one thing from any other thing impossible. The only connection between the Electric Monk and these clones are 1) the color pink, and 2) the authors had a copy of this book with them during the long drive to Yellowstone for their field work. Informal strain designations can come from anything - the students initials, their cats name, love interest, or a sports team - anything.