The detection & identification of unculturable organisms

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In a typical molecular phylogenetic analysis, such as those we're doing in lab, you start wih genomic DNA isolated from a pure culture an organism. The ssu-rRNA sequence obtained by PCR amplification is used to determine the place of that organism in the 'big tree' - it's "phylotype".

It is also possible to amplify rDNA genes from a population of organisms. In this case, the PCR product is a mixture of sequences, and so it is generally necessary to clone the products before sequencing, so that each sequencing reaction contains a single sequence. Is is common, afterwards, to design specific fluorescently-labeled oligonucleotide probes based on these sequences to identify the organisms (by FISH) that each sequence represents in the original population. This FISH analysis can be used to confirm the identity of any specific organism in the population, to determine the morphology of a specific organism, or as a handle on the organism for attempts to grow the organism in culture.

the Search

Some examples of uncultivated species characterized by molecular phylogenetic analysis:

  • The identification (and ultimately cultivation) of the pink filamentous organism that grows in the outflows of neuttral pH hot springs in Yellowstone National Park. Initial attempts to grow this organism resulted, in the 1960's, in the cultivation instead of an entirely different pink bacterium - Thermus aquaticus.

  • The sulfur-oxiding symbionts of the giant tube worm, the vent clam, and the scaly snail. These organisms are found only at deep-sea hydrothermal vents, and have no digestive tracts - they are feed entirely by their sulfur-oxidizing proteobacterial endosymbionts. A similar example is the endosymbiont of the scaly snail.

  • Magnetotactic Bacteria - can be isolated by taking advantage of their magnetotaxis, but cannot be grown in culture.

  • Bacillary angiomatosis - a disease that turns out to be caused by a close relative of the causative agent of cat scratch disease, Rochalimaea quintana. This knowledge, obtained by molecular phylogenetic analysis, has allowed the cultivation of the organism, now known as Bartonella bacilliformis, using culture methods used for Rochalimaea.

  • Cenarchaeum symbiosum is a psychrophilic (grows optimally at 10C) crenarchaeon symbiont of an unnamed marine sponge of the genus Axinella. They (the symbionts) are related in ssu-rRNA sequence to abundant crenarchaeal species found in marine and soil environments, although none have been cultivated.

  • Epulopiscium fishelsonii, for a long time the largest known prokaryote, is a gut symbiont of some marine herbivorous fish (surgeonfish, doctorfish, & tangs), was originally thought to be a protist, perhaps a ciliate, based on size (big enough to see clearly with the naked eye), but didn't seem to have any organelles. It turns out to be a Firmicute!

  • Sap-sucking and wood-eating insects contain an unusual organ, called the bacteriome, consisting of about 75 cells called bacteriocytes. These cells contain endosymbiotic oval bacteria, that provide the insect with the vitamins and essential amino aids that are absent in their nutritionally poor diets. This is an obligate relationship - the insect cannot survive without the bacteria (antibiotic treatment is lethal), and the bacteria have never been cultivated outside of the insect. However, they have been characterized using ssu-rRNA analysis - most are gamma purple Bacteria, but some are beta-purples and some are Cytophagales. One such insect endosymbiont, Buchnera aphidicola, has had it's entire genome sequenced even though it can't be cultivated except in aphids.

And many, many, more....