Cultivation of the YNP pink filaments organism : Thermocrinus ruber

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pdfHuber R, et al. 1998 Thermocrinus ruber, gen. nov., sp. nov., a pink-filament-forming hyperthemophilic bacterium isolated from Yellowstone National Park. Appl. Env. Microbiol. 64:3576-3583

The task: Cultivate the YNP pink filamentous organisms (EM17) on the basis of its phylotype, and isolate it as a pure culture from a single cell.

In 1994, the phylotype of the pink filamentous organism common in Yellowstone National Park neutral pH hot springs had been determined; it was closely related to Aquifex. In the 4 years since, additional genera of organisms in this group had been identified,Hydrogenobacer and calderobacterium. All of the cultivated members of this phylogenetic group are physiologically similar, being thermophilic hydrogen oxiders. So, in attempt to cultivate the pink filamentous organism EM 17, a series of enrichment cultures based on the known conditions in Octopus Spring and the optimal growth conditions for growth of Aquifex were made. The chemical composition of Octopus Spring was mimicked in detail, and provided with both hydrogen and oxygen in low concentrations of 3% each. These enrichments were innoculated with washed pink filaments from Octopus Spring.

The progress of the enrichment cultures was assessed by microscopic examination (growth of pink filaments) and by testing samples by FISH for hybridization with the EM17 probe. Although no cultures resulted in pink filamentous growth, some enrichments yielded good growth of a rod-shaped pink bacterium that hybridized strongly to the EM17-specific probe.

In order to isolate this organism in pure culture, a 1064nm (about 1 micron) wavelength focused infrared laser was used as 'optical tweezers' to capture single cells that looked like the ones that hybridize to the EM17-specific probe. Note that FISH analysis kills cells (you fix them in glutaraldehyde), so you can only test cultures after the fact, you can't test the individual cells before separating them out and growing them.

optical tweezers

These cells were injected into fresh culture media for growth. Of course, many cells were collected and cultured in this way, and the resulting growth, if any, was tested using the EM17 probe. Some of these cultures DID grow, and when tested one (at least) strongly hybridized to the EM17-specific probe.

With a pure culture in hand, the ssu-rRNA sequence of this organism was determined and found to be nearly identical to the EM17 sequence.

Phylogenetic tree of EM17 compared to the newly-isolated organism "Thermocrinus ruber". (Fig. 7 in Huber 1998 AEM 64:3576)

...although not exactly the same. This is typical - most microbial populations are collections of closely-related organisms, specialized for different micro-environments. Enrichment cultures typically capture only the fastest-growing, robust types (i.e. the weeds), whether they are the predominant strain or not (usually not). The organism was named Thermocrinus ruber ("hot red hair").

Thermocrinus ruber grows as long rods in suspension cultures, but when cultivated in media prepared to match the composition of Octopus Spring water in an artificial 'creek', with a turbulent overflow, grows as nice pink filaments that are just like those seen in the wild.

T. ruber cells grown in suspension culture (Fig 2 in Huber 1998 AEM 64:3576)

T. ruber
cells grown in suspension culture (Fig 4 in Huber 1998 AEM 64:3576)

T. ruber cells grown in the articial creek environment (Fig 3 in Huber 1998 AEM 64:3576)

articial Octopus
Schematic of the artifical hot spring overflow culture container using to grow T. ruber as filaments (Fig 1 in Huber 1998 AEM 64:3576)

This paper is a monogram - a formal description and naming of a newly isolated organism, and at the end of the paper, you'll see a series of more-or-less standard tests used to describe the general properties of the organism; the sort of data you'd find in Bergy's Manual, for example.