More Sand Grains

by Christoph

What you see here in Fig­ure 1d com­petes with a NASA photo from 2012 for the best shot of the Milky Way but – spoiler alert! – it's ac­tu­ally a close-up of some hum­ble mi­crobes gath­ered on a grain of sand (an­other one is on the front­page ). I con­cluded the pre­vi­ous post about bac­te­ria on sand grains with the ques­tion: and where are the ar­chea ? The rea­son for this ques­tion wasn't sheer cu­rios­ity: based on a sur­vey of prokary­otic di­ver­sity in the sub­sur­fa­ce wa­ter of the Pa­cific Ocean, Karner et al.  es­ti­mated  that the global oceans are pop­u­lated by 1.3 × 10²⁸ ar­chaea and 3.1 × 10²⁸ bac­te­ria (in­clud­ing an es­ti­mated 2.4 × 10²⁸ SAR11/ Pelag­ibac­ter ubique cells ). No, I won't jug­gle with num­bers here again. But if ar­chaea do in­deed rep­re­sent ~1/3 of the prokary­otes in the oceans it would be sur­pri­sing to not find them at least in the up­per lay­ers of the sed­i­ments, on sand grains for ex­am­ple. Well, here they are, see Fig­ure 1b.

Fig­ure 1.  Di­rect vi­su­al­iza­tion of taxa on sand grains us­ing CARD-FISH and con­fo­cal laser scan­ning micro­scopy. Tar­geted taxa are in­di­cated in the in­di­vid­ual pan­els b, d, and e (front­page). DA­PI sig­nal (in blue) shows all cells not tar­geted by the probes. Im­ages are com­pos­ite mi­cro­graphs of flu­o­res­cent sig­nals and trans­mit­ted light of the sand grain's sur­face. Micro­graph b is a su­perresolution struc­tured il­lu­mi­na­tion im­age (SR-SIM). If not oth­er­wise in­di­cated, scale bar refers to 10 μm. Source (Open Ac­cess PDF here)

When Probandt et al. made their 'cen­sus' of mi­crobes on sin­gle sand grains in a sam­ple of North Sea sedi­ment they found up to 10⁵ mi­cro­bial cells per grain, and these were, in their ma­jor­ity, mem­bers of 10 bac­te­r­ial taxa. To also de­tect ar­chaea they em­ployed CARD-FISH, a tech­nique that al­lows to de­tect mi­crobes from phy­lo­ge­net­i­cally di­verse clades di­rectly in their nat­ural habi­tat (CARD-FISH is briefly de­scribed in the leg­end for Fig­ure 2 ). Ac­cord­ing to their oligonu­cleotide pro­be, the sin­gle cells and small ag­gre­gates of ar­chaea (3 – 10 cells; green dots in Fig­ure 1b ) be­long to the Thau­marcheota, a phy­lum of the ar­chaea that was for­merly 'lumped to­gether' with the Cren­ar­cheota, and whose known mem­ber species are chemolithoau­totrophic am­mo­nia-ox­i­diz­ers. Sus­piciously, these ar­chaea were some­times found in close neigh­bor­hood (0 – 10 µm ) of small ag­gre­gates of Ni­tro­spi­rae bac­te­ria (red dots ). 'Sus­pi­cously' be­cause it is well known that ar­chaeal ammo­nia ox­i­diz­ers (AOA) and their bac­te­r­ial coun­ter­parts from the phyla Ni­tro­spi­rae and Beta­pro­teo­bac­te­ria (AOB) usu­ally oc­cur as densely clus­tered com­mu­ni­ties, for ex­am­ple in waste wa­ter treat­ment plants (with the ar­chaea ex­ceed­ing the bac­te­ria in cell num­bers by a fac­tor of 10 ). The co-lo­­cal­iza­tion of ar­chaeal and bac­te­r­ial am­mo­nia-ox­i­diz­ers on the sand grains in­di­cates that the en­tire ni­tro­gen cy­cle can take place in the up­per, per­me­able layer of seafloor sed­i­ments where meta­bolic in­ter­me­di­ates can be trans­ported ad­vec­tively through the ma­trix.

Fig­ure 2. Setup of a CARD-FISH ('Cat­alyzed Re­por­ter De­po­si­tion Flu­o­res­cence in situ Hybri­di­za­tion') expe­ri­ment. Prokary­otic cells in so­lu­tion or in situ are per­meabilized by treat­ment with lysozyme and/or achro­mopeptidase. Step 1: af­ter in­ac­ti­va­tion of en­doge­nous per­ox­i­dases, the per­me­abi­lized cells are in­cu­bated with rRNA-tar­get­ing oligonu­cleotides for hy­bridiza­tion; the oligonu­cleotides are cou­pled to horse­rad­ish per­oxidase (HRP). Step 2: The cells are incu­ba­ted with flu­o­rophore-cou­pled tyra­mide in the pres­ence of H2O2 HRP cou­pled to the hybridi­zed oligonu­cleotides cat­alyzes lo­cally a re­ac­tion that re­sults in the pro­duc­tion of hy­droxyl radi­cals (·OH) from H2O2 in the pres­ence of O2·^–. The ra­dicalized tyra­mine now binds co­va­lently to nearby pro­tein mol­e­cules, thereby amplify­ing the sig­nal (up to 100-fold). The read­out is done with a con­fo­cal laser mi­cro­scope. Modi­fied from Source

By com­bin­ing metage­nomics with cell iden­ti­fi­ca­tion by lo­cal­iza­tion – thus al­low­ing the re­con­struc­tion of mi­cro­bial com­mu­ni­ties and their meta­bolic path­ways – the study by Probandt et al. is an ex­cel­lent ex­am­ple for the res­o­lu­tion power of the cul­ture-in­de­pen­dent charac­te­ri­za­tion of a par­tic­u­lar mi­cro­bial habi­tat. Add to this meta­transcriptomics (a word­mon­ster, mean­ing that reg­u­la­tion and ex­pres­sion pro­files are gen­er­ated in ad­di­tion to and for pro­jec­tion onto metage­nomic data ) and you can ac­cess not only ques­tions like "who's there?", and "who's where, ex­actly?", but also "who's busy and thriv­ing? who's chil­ling?". This doesn't sound a lot like an ap­proach that could fit into the straight­jacket of Koch's pos­tu­lates but, hey, study­ing mi­cro­bial con­sor­tia in the wild is more fun. Ah! Be­fore I for­get: there are also eu­kary­otic mi­crobes, and they also pop­u­late the sand grains. Based on 16S rRNA se­quences Probandt et al. clas­si­fied them as al­gal chlo­roplasts, and based on mor­phol­ogy as (mostly ) dia­toms with a high species di­ver­sity. They were also detec­ted by chloro­plast aut­o­flu­o­res­cence and a Eu­karya-spe­­ci­fic probe in CARD-FISH ex­per­i­ments.

An aside: sand grains had al­ready at­tracted the ever-cu­ri­ous An­tonie van Leeuwen­hoek (1632–1723), and he in­cluded a draw­ing of sand grains as he saw them through his mi­cro­scope in a let­ter to the Royal So­ci­ety, 4th Decem­ber 1703. No, he did not see his cher­ished an­i­mal­cules on the sand grains.