One Grain of Sand

by Christoph

Mi­cro­bi­ol­o­gists face three prob­lems to­day when talk­ing to fel­low bi­ol­o­gists or any cu­ri­ous audi­ence: the num­bers of prokary­otes and uni­cel­lu­lar eu­kary­otes, their di­ver­sity, and their tini­ness (ti­ny in their ma­jor­ity, for no­table ex­cep­tions see here and here in STC ). Sure, one can do the maths and jug­gle with num­bers. That's en­ter­tain­ing, and every­body is amazed when they learn that mi­cro­biologists eas­ily count up to 10⁸ in­di­vid­ual cells in an E. coli  colony with a di­am­e­ter of ~1.5 mm on an LB-medium agar plate. As in­di­vid­u­als, that's roughly equiv­a­lent to the pop­u­la­tion of the Phi­lippines, #12 on the Wikipedia list of the most pop­u­lous coun­tries. But mi­cro­bi­ol­o­gists strug­gle no less than ge­ol­o­gists, geo­chemists, or lay peo­ple with com­pre­hend­ing what the es­ti­ma­ted 2.4 × 10²⁸ SAR11/Pelag­ibac­ter ubique  cells in the world's oceans mean for, say, Earth's car­bon cy­cle (they are prob­a­bly the most abun­dant of the small things, con­sid­ered here by Merry ). Or take the Pro­chlo­ro­coccus  fed­er­a­tion with an es­ti­mated ~10²⁷ cells in the oceans, which is, to­gether with the en­tire pho­to­syn­thetic pi­coplank­ton, re­spon­si­ble for the pro­duc­tion of ~50% of the at­mos­pheric oxy­gen on our planet.

Jug­gling with num­bers is the fun part, but what's com­pletely burst­ing the bounds of imag­i­na­tion is the di­ver­sity of the prokary­otes as re­vealed dur­ing the last decade by metage­nomics. Con­sider, for a mo­ment, that a ba­nana and a chimp are ge­net­i­cally more sim­i­lar to each other than the above men­tioned Pelag­ibac­ter  and Prochloro­coc­cus. Ba­nana and chimp Rad51 pro­teins, for ex­am­ple, are 70% iden­ti­cal while the Rad51 or­thologs in bac­te­ria, RecA, of Pelag­ibac­ter  and Pro­chlo­ro­coc­cus  are only 58% iden­ti­cal (though still shar­ing ~40% iden­tity with Rad51 of ba­nana and chimp, or Ho­mo sapi­ens for that mat­ter ). Both bac­te­r­ial species be­long, like ba­nana and chimp, to dif­fer­ent ta­xonomic phyla, of which we presently have 100+ for the prokary­otes. And, just last month, Parks et al. added a whop­ping ~8,000 bac­te­r­ial and ar­chaeal genomes as­sem­bled from metage­nomic da­ta to the 'mas­ter col­lec­tion' (as of De­cem­ber 2017, NCBI lists 125,700 se­quenced prokary­otic geno­mes; many are still in­com­plete and there's also a great deal of re­dun­dancy, for ex­am­ple >200  H. py­lori genomes ). Thus we do not only have many di­verse phyla but these are pop­u­lated by vast num­bers of dif­fer­ent ex­tant species (this de­spite the on­go­ing de­bate of what con­sti­tutes a 'bac­te­r­ial species', and al­though the phyla are prob­a­bly not evenly pop­u­lated; you can get an im­pres­sion here).

Fig­ure 1. Mi­cro­bial col­o­niza­tion of a sand grain. Con­focal laser scan­ning micro­graph show­ing SY­BR green I‑stained mi­crobial cells on a sand grain vi­su­al­ized as three-di­men­sional re­con­struct­ion. The grain's sur­face was vi­su­al­ized by trans­mitted light mi­croscopy. Note the bare sur­fac­es of con­vex and ex­posed ar­eas in con­trast to pro­tected ar­eas dom­i­nated by macro­to­­po­gra­phy, which are densely po­pulated by mi­crobes. Bar: 100 µm. Source (Suppl. Ma­te­r­ial; Open Ac­cess PDF here). Front­page: binoc­u­lar photo­graph of dried sand. Bar: 1 mm. Source (Suppl. Ma­te­r­ial).

But be­fore you feel drowned in num­bers, here's some­thing more tan­gi­ble. Some­thing so small that it is close to the limit of be­ing vis­i­ble to the naked eye but still per­cep­tible when you take it be­tween thumb and in­dex fin­ger: a grain of sand in the sub-mil­lime­ter size range (Krum­bein phi val­ues of 1 – 2, or 'medium sand' in the terms of gra­nulometry, see fron­tispiece). Ka­trin Knit­tel and her co-work­ers at the MPI for Ma­rine Mi­cro­bi­ol­ogy in Bre­men, Ger­many, dis­cuss in a re­cent study their 'pop­u­la­tion cen­sus' of the sand grain shown in Fig­ure 1 and of 17 addi­tio­nal sin­gle sand grains (SSGs 1 – 17, por­trayed in the top row of Fig. 2 ). They had picked these sand grains from push core sam­ples of the first 2 cm of the sub­ti­dal sedi­ment at a depth of 8 m on a site within the shal­low 'Hel­go­land Roads' just off the coast of the is­land of Hel­goland in the North Sea (I did not know be­fore that the 'Hel­goland Roads' – not the usual 'roads' for car traf­fic here but the mar­itime term for an an­chor­age out­side a har­bor – are mon­i­tored by ma­rine bi­ol­o­gists since 1962 on a daily ba­sis, while plank­ton re­search at this site ac­tu­ally dates back to the end of the 19th cen­tury ).

The sand grain you see in Fig­ure 1 is densely pop­u­lated by mi­crobes. To vi­su­al­ize those mi­crobes, Probandt et al. stained their nu­cleic acids with SYBR-Green I (stains pref­er­en­tially ds­DNA, less so ss­DNA and RNA ) in a so­lu­tion that also con­tained Mowiol 4–88 (a polyvinyl al­co­hol used to em­bed his­to­log­i­cal sam­ples ). Af­ter hard­en­ing of the poly­mer (keep­ing any at­tached mi­crobes in place ), the grain was scanned with a con­fo­cal laser mi­cro­scope for flu­o­res­cence, and, si­mul­ta­ne­ously, for its sur­face topol­ogy with a trans­mit­ted-light mi­cro­scope. What you then see in Fig­ure 1 is a three-di­men­sional re­con­struc­tion from both data sets. Ob­vi­ously, mi­crobes do not pop­u­late the sand grain evenly but clus­ter in in­dents and cav­i­ties while avoid­ing ex­posed con­vex ar­eas (a fact known since at least 1968 ). The au­thors found sim­i­lar bi­ased dis­tri­b­u­tion on other grains but they are not sure whether it is rather a con­se­quences of grains rub­bing with each other dur­ing tur­bu­lent move­ments of the up­per sed­i­ment layer thus dis­lodg­ing mi­crobes by shear forces (shown ear­lier to oc­cur read­ily in an ex­per­i­men­tal setup ) or whether grow­ing in in­dents and cav­i­ties shel­ters mi­crobes from graz­ing by larger eu­kary­otic preda­tors. In any case, it's a sign for on­go­ing drama on the sea­floor.

Fig­ure 2. Com­po­si­tion of bac­te­r­ial com­mu­ni­ties on sin­gle sand grains and in bulk sed­i­ment as shown by Il­lu­mina par­tial 16S rRNA gene se­quen­cing. De­picted are the 10 most se­quence-abun­dant fam­ily-level clades found on the indi­vi­dual sand grains or in bulk sed­i­ment da­tasets. Re­main­ing fam­ily level clades are sum­ma­rized as "other". Clades de­scribed as "un­cul­tured (un­cult)" could not be fur­ther clas­si­fied and might con­tain nume­rous OTU_0.97 of re­spec­tive taxon level. De­picted com­munity com­po­si­tion is bas­ed on sub­sam­pled datasets (N=44,901). * Se­quen­ces clas­si­fied as Planctomyce­ta­ceae and Phy­cis­phaer­aceae rather com­prise sev­eral un­classified fam­i­lies within the class Planc­to­my­cetia and Phyci­sphaerae, re­spec­tively. Source (Suppl. Ma­terial; Open Ac­cess PDF here)

A closer in­spec­tion of more sand grains re­vealed all com­mon mi­cro­bial mor­pho­types such as short and long rods, coc­ci as well as fil­a­ments, which grew mostly in mo­nolay­ers with, as eval­u­ated by soft­ware-sup­ported measure­ments, cell-to-cell dis­tances in the lower mi­crom­e­ter ran­ge, that is, vir­tu­ally side-by-side like in biofilms (al­ready seen in scan­ning elec­tron mi­cro­graphs of bac­te­ria attach­ed to sand grains from 1978, here's an ex­am­ple; note that at that time biofilm was called 'poly­mers', the term 'biofilm' be­came pop­u­lar only in the 80s ). As con­trol for the soft­ware-aided in situ  counts they per­formed 'to­tal cell counts' by dis­lodg­ing cells from the grains by sev­eral rounds of ul­tra­son­i­ca­tion, pu­ri­fy­ing the su­per­natants by fil­tra­tion, stain­ing them with acri­dine or­ange (a DNA/RNA stain ) and count­ing the stained cells un­der an epi­fluor­es­cen­ce mi­cro­scope. The au­thors do not men­tion it expli­cit­ly but it seems that acri­dine-stained cells gave more re­li­able cell counts while the SYBR Green‑stained cells al­lowed safe state­ments about cell-to-cell dis­tances and about pref­er­en­tial cell clus­ter lo­cal­iza­tions that re­flect the topol­ogy of the grains. I men­tion this to il­lus­trate that rarely a sin­gle method gives the ex­per­i­menter all the wanted an­swers.

For one set of 17 sand grains, Probandt et al. asked the mi­crobial pop­u­la­tions of each grain for their 'ID cards', that is, they PCR-am­pli­fied a par­tic­u­lar stretch of 16S rDNA (=the stretch of chro­mo­so­mal DNA that en­codes the 16S ri­bo­so­mal RNA ) di­rectly from the grains by ap­pro­pri­ate primers. You can take that quite lit­er­ally, as they write:"Grains were ran­domly picked with ster­ile, DNA-free for­ceps, size and ap­pear­ance doc­u­mented by pho­tographs and trans­ferred into PCR strips (one grain per re­act­ion) filled with ster­ile wa­ter. The pro­to­col for SSG-PCR was as de­scribed for bulk sed­i­ments. For each grain, new for­ceps were used and dipped into a sep­a­rate PCR re­ac­tion mix as a neg­a­tive con­trol be­fore pick­ing the grain. There was no PCR prod­uct in any of the neg­a­tive con­trols." The PCR prod­ucts were bulk‑sequenced and, as is com­mon among bioin­for­mati­cians, the ob­tained se­quen­ces grouped in OTUs, that is, 'op­er­a­tional tax­o­nomic units' used as proxy for species when apply­ing a 97% cut-off (OTU_0.97). They could thus as­sess the ap­prox­i­mate dis­tri­b­u­tion of higher-or­der taxa (phy­lum, class, or­der, fam­ily ) and species rich­ness in the col­lec­tion of their se­quence data, that is, from un­cul­ti­vated bac­te­ria and ar­chaea (Fig­ure 2). The au­thors con­cluded: "...each sand grain in­ves­ti­gated in this study was the habi­tat for around 10⁵ cells rep­re­sent­ing sev­eral thou­sand species. The av­er­age dis­tance be­tween any two cells on a sand grain in pro­tected ar­eas was 0.5±0.7 μm and there­fore 100-fold shorter than the av­er­age dis­tance be­tween cells in the wa­ter col­umn." The 10 most se­quence-abun­dant fam­ily-level clades were the same on all sand grains, and not grossly dif­fer­ent from those in bulk sedi­ment sam­ples. How­ever, the Planc­to­mycetes (see Daniel's post on the PVC phy­lum here in STC ) were more nu­mer­ous and more dif­fer­en­ti­ated in bulk sedi­ment while the Woe­seiaceae ap­par­ently pop­u­lated the grains with more. Woe­seiaceae is a lit­tle stud­ied fam­ily (just 3 PubMed en­tries ) within the Chro­ma­tiales, an or­der of the Gamma­pro­teo­bac­te­ria in which many mem­bers are pho­to­syn­thet­i­cally ac­tive yet breath sul­fur in­stead of oxy­gen (for­merly, they were called 'pur­ple sul­fur bac­te­ria' ). They sum­ma­rize their find­ings:"In con­trast to sta­ble soils, the dy­namic and per­me­able sur­face sed­i­ments are char­ac­ter­ized by fluc­tu­at­ing re­dox con­di­tions and sub­strate avail­abil­i­ties re­sult­ing in di­verse and ver­sa­tile mi­cro­bial com­mu­ni­ties. Our data sug­gest that each sand grain is a small mi­cro­bial repos­i­tory from which the ma­jor cy­cles of car­bon, ni­tro­gen and sul­fur trans­for­ma­tions typ­i­cal of ma­rine ben­thic habi­tats could be re­con­sti­tu­ted." There you have it, a world on a grain of sand.

I should men­tion that Probandt et al. did not make a capri­cious trip from Bre­men to Hel­goland only to col­lect a spoon­ful of sand grains for analy­sis (3 h by ship from Bre­mer­haven, no big deal ). The 'sand grains' where part of their in-depth analy­sis of mi­cro­bial com­mu­ni­ties in sub­lit­toral sur­face sed­i­ments and bot­tom wa­ter at sev­eral sites in the North Sea (at dis­tances of up to ~162 nmi [nau­ti­cal miles] from Hel­goland, that is, roughly 300 km ). They found in this sur­vey a fairly sim­i­lar com­po­si­tion of bac­te­r­ial taxa in these sed­i­ments as on the sand grains from the Hel­goland Roads, and in the re­spec­tive bot­tom wa­ter sam­ples a preva­lence of (no sur­prise, see first para­graph) SAR11/ Pelag­ibac­ter ubique, which was vir­tu­ally ab­sent in the sed­i­ments (you can peek at their sum­ma­riz­ing ta­ble here ). They did ap­par­ently not run into the 'ma­rine snow trap' (men­tioned by Roberto here in STC ), that is, slowly set­tling or­ganic de­bris – in­clud­ing DNA – from dead or­gan­isms in the wa­ter col­umn ob­scur­ing re­sults from sed­i­ment analy­sis. The num­bers of cyanobac­te­ria were low in the sed­i­ments and in the bot­tom wa­ter sam­ples at any but one of the sites, which is un­sur­pris­ing as – to come full cir­cle – Prochloro­coc­cus doesn't thrive in northerly lat­i­tudes above ~40°.

You won­der whether there weren't any ar­chaea on those sand grains ? There were, of course, and I will soon come back to them in a fol­low-up post.