A Fly in the Frozen Cus­tard

by Elio

Don't you just love a good con­tro­versy? Take, for in­stance, the dis­cus­sions on long term sur­vival of bac­te­ria. De­pend­ing on your point of view, bac­te­ria ex­ist in salt for­ma­tions go­ing back 250 mil­lion years, in 50 mil­lion year old am­ber, or in 500,00 year old per­ma­nently frozen ice or per­mafrost. Some re­searchers re­port the iso­la­tion of vi­able bac­te­ria, oth­ers are con­tent with their DNA (usu­ally in pieces). Find­ing some­thing in­ter­est­ing turns out to be rel­a­tively easy; prov­ing that it orig­i­nated when you say it did, less so. The far­ther back you go, the more con­tentious the is­sue be­comes. But in­creased experi­mental so­phis­ti­ca­tion is be­ing brought to bear and the con­clu­sions about long-term sur­vival are be­com­ing more pointed.

Glacial ice in Antarc­tica. Source

A cher­ished no­tion has been that the sur­viv­ing cells are in some form of dor­mancy. Surely what comes to mind are spores, those mas­ters of re­sis­tance, no?

This thought has come un­der scrutiny. For starters, bac­te­ria sur­viv­ing in an­cient ice for­ma­tions are not all spore­form­ers. Fur­ther­more, per­haps dor­mancy is not suf­fi­cient. Maybe sur­vival de­pends on a small mea­sure of ac­tive me­tab­o­lism, enough to sup­port DNA re­pair. Think about it. A spore may be re­sis­tant to a wealth of phys­i­cal chal­lenges, but its DNA is still vul­ner­a­ble to nat­ural ra­dia­tion. In­deed, spore­form­ers lack ac­tive DNA-re­pair ma­chin­ery, but this is a generic prob­lem, true for any cell that en­dures for a long time. So, the no­tion that DNA re­pair takes place pari passu with dam­age is ap­peal­ing.

A group of sci­en­tists from no less that 6 dif­fer­ent coun­tries (Aus­tralia, Canada, Den­mark, Rus­sia, Swe­den, USA) came up with the al­ter­na­tive to dor­mancy. They an­a­lyzed sam­ples of con­tin­u­ously frozen per­mafrost, some go­ing back 500,000 years, and con­cluded that the bugs therein are ac­tually ac­tive, al­beit at a very slow rate of me­tab­o­lism. Isn't this a neat way of think­ing about the prob­lem of long term sur­vival?

To de­ter­mine whether those bugs had been re­pair­ing their DNA all those years, the au­thors used a clever ex­per­i­men­tal strat­egy based on a re­ac­tion that pro­ceeds at a rel­a­tively slow rate: the spon­ta­neous deam­i­na­tion of cy­to­sine in DNA to uracil. Ac­tive cells can re­pair this dam­age, replac­ing the uracil with cy­to­sine. How can you tell whether the uracils have ac­cu­mu­lated or been re­paired? Treat the DNA with uracil-N-gly­co­sy­lase, an en­zyme that breaks the ri­bose-uracil bond. This frag­ments DNA that con­tains un­re­paired uracils but leaves in­tact the re­paired DNA. Long (4 kb) am­pli­cons can only ob­tained from DNA of meta­bol­i­cally ac­tive cells be­cause uracil-con­tain­ing un­re­paired DNA will be cut into smaller frag­ments.

Col­wellia psy­chrery­thraea, a model cold-adapted or­gan­ism. Source

The au­thors started with sam­ples of DNA re­cov­ered from per­mafrost and Antarc­tic ice of vary­ing age, rang­ing from mod­ern up to 1 mil­lion years old. Fol­low­ing treat­ment with uracil-N-gly­co­sy­lase, they PCR-am­pli­fied 4 kb frag­ments that in­cluded por­tions of 16S and 23S ri­bo­so­mal RNA genes as well as the ITS re­gion. With the base se­quences in hand, they could iden­tify the or­gan­isms pre­sent in sam­ples of vary­ing ages. In the old­est sam­ples, the dom­i­nant bac­te­ria were mem­bers of the high-GC Gram-pos­i­tive Acti­nobac­te­ria largely re­lated to the non­sporeforming Arthrobac­ter. The amount of in­tact DNA re­cov­ered from spore­form­ers, on the other hand, de­clined with time.

The au­thors also showed that sam­ples up to about 600,000 years of age evolved CO₂ while frozen at −10 °C un­der anaer­o­bic con­di­tions for some 9 months. This sug­gests that even when frozen, some slow me­tab­o­lism con­tin­ues. Ergo, DNA re­pair may be pos­si­ble.

Wow! Look at this! This phage thinks Col­wellia psy­chrery­thraea is meta­bol­i­cally ac­tive. Source

There is, how­ever, a fly in the frozen cus­tard. A thought­ful pa­per by sev­eral au­thors at North­east­ern Uni­ver­sity in Boston brings up the ob­ser­va­tion that old ice is not all that uni­form, but is rid­dled with chan­nels filled with li­quid wa­ter. Ac­tive me­tab­o­lism is likely here. Some psy­chrophilic bac­te­ria such as Col­wellia psy­chrery­thraea, are ca­pa­ble of swim­ming at high speeds at −10°C; oth­ers are ca­pa­ble of di­vid­ing every 160 days in per­mafrost sam­ples main­tained at −20 °C in the lab. Fur­ther­more, some bac­teria ap­pear to have phys­i­o­log­i­cal mech­a­nisms that en­able them to adapt to very cold con­di­tions. Is it not pos­sible then, that the bac­te­ria found in per­mafrost or gla­cial ice may not be all that an­cient? That they are alive and well and go­ing about their busi­ness at a re­spectable pace in your frozen cus­tard?