The Most Abun­dant Small Things Con­sid­ered

by Merry Youle & Gemma Reguera

The pi­o­neer­ing se­quenc­ing of en­vi­ron­men­tal 16S rRNA bac­te­r­ial genes from Sar­gasso Sea sam­ples, re­ported by Gio­van­noni and col­leagues in a 1990 Na­ture pa­per, re­vealed a great ge­netic di­ver­sity of ma­rine bac­te­ria. It also iden­ti­fied the most abun­dant rRNA se­quences as be­long­ing to a group of bac­te­ria with no cul­ti­vated rep­re­sen­ta­tives, a group col­lec­tively known as the SAR11 clade. Sub­se­quent quan­ti­ta­tive stud­ies pro­vided ev­i­dence for the as­ton­ish­ing num­bers that SAR11 cells reach in the oceans. On av­er­age, the SAR11 clade com­prised one third of all of the prokary­otic cells in the sur­face wa­ters (the eu­photic zone), ac­count­ing for up to 50% of the to­tal in some re­gions. The clade was also abun­dant in deeper lay­ers (the sube­uphotic zone be­low 200 m), be­ing on av­er­age one fifth of the cells here and, in some cases, rep­re­sent­ing up to 25% of the mi­cro­bial com­mu­nity. It is easy to ap­pre­ci­ate their dom­i­nance in terms of cell num­bers per ml of wa­ter: 2.0 × 10⁵ ml⁻¹ in the eu­photic zone and 0.2 × 10⁵ ml⁻¹ in the wa­ters be­low. Not only are they abun­dant, they are ubiq­ui­tous. Global es­ti­mates are that there are 2.4 × 10²⁸ SAR11 cells in the oceans!

Fig­ure 1. Pelag­ibac­ter ubique. Credit: Ke­hau Manoi; C‑MORE, Hawai'i. Source

De­spite their abun­dance and global dis­tri­b­u­tion, this enig­matic SAR11 clade de­fied cul­ti­va­tion un­til 2002, when the first rep­re­sen­ta­tive, Pelag­ibac­ter ubique strain HTCC1062, was cul­tured. In con­trast to stan­dard prac­tices that rely on rich cul­ti­va­tion me­dia for iso­la­tion of novel strains, the re­searchers used very low nu­tri­ent con­cen­tra­tions, ster­il­ized nat­ural sea­wa­ter, and other con­di­tions that closely mim­ic­ked the nat­ural habi­tat. Close at­ten­tion to the en­vi­ron­men­tal vari­ables paid off and al­lowed for the en­rich­ment of SAR11 cells in the cul­tures and the suc­cess­ful iso­la­tion of sev­eral rep­re­sen­ta­tive strains. The re­searchers were also pa­tient: gen­er­a­tion times for the strains ranged from 25 to 50 h. Slow as this may be by lab­o­ra­tory stan­dards, it is sim­i­lar to or even faster than the over­all growth rates mea­sured for the to­tal mi­cro­bial com­mu­ni­ties of which SAR11 cells are a part. Fur­ther­more, the con­tri­bu­tion of the SAR11 cells to het­erotrophic pro­duc­tion is greater than ex­pected based on their rel­a­tive abun­dance, par­tic­u­larly when phos­phate is lim­it­ing. For ex­am­ple, SAR11 bac­te­ria from the North­west At­lantic Ocean, which ac­count for 25% of the mi­cro­bial com­mu­nity in these aquatic en­vi­ron­ments, can in­cor­po­rate more than half of the amino acids and glu­cose as­sim­i­lated by the mi­cro­bial com­mu­nity as a whole. Not bad!

Fig­ure 2. Red marks in­di­cate lo­ca­tions where SAR11 rRNA genes have been de­tected us­ing mol­e­c­u­lar tech­niques. Al­though 52 in­di­vid­ual sam­ples are rep­re­sented, many stud­ies along the US Pa­cific coast and in the North At­lantic have been con­ducted at the same sam­ple lo­ca­tions and are there­fore rep­re­sented by a sin­gle red mark. Source

Small and "gassy"   Hav­ing P. ubique in cul­ture al­lowed the Gio­van­noni lab to start in­ves­ti­gat­ing the strate­gies that ac­count for its ex­tra­or­di­nary suc­cess. The first no­table char­ac­ter­is­tic is that these bac­te­ria are small. Soon af­ter cell di­vi­sion, cells are cres­cent-shaped, 0.37 – 0.89 μm long with a di­am­e­ter of 0.12 – 0.20 μm. Such di­men­sions yield a vol­ume of ~0.01 μm³, mak­ing P. ubique one of the small­est free-liv­ing cells ever cul­tured. For com­par­i­son, 'tiny' au­totrophic Prochloro­coc­cus has a 0.6 μm di­am­e­ter, and the di­am­e­ter of the small­est eu­kary­ote presently known, Os­tre­o­coc­cus tauri, is 1 μm. By com­bin­ing this small vol­ume with a vib­ri­oid shape, P. ubique achieves an even higher sur­face-to-vol­ume ra­tio, which is crit­i­cal for max­i­miz­ing the flux of nu­tri­ents and waste prod­ucts into and out of the cell, re­spec­tively. In­creased dif­fu­sional rates con­fer an ad­van­tage on these small cells when scav­eng­ing the scarce nu­tri­ents avail­able in their olig­otrophic en­vi­ron­ments, thus pro­mote their growth. Not sur­pris­ingly, de­spite their small size, en masse they rep­re­sent 12% of the ma­rine prokary­otic bio­mass.

Their ubiq­uity, abun­dance, and ef­fi­cient meta­bolic adap­ta­tion to their en­vi­ron­ment sug­gest that these bac­te­ria have a ma­jor im­pact on global car­bon cy­cling. SAR11 cells live and grow by con­vert­ing la­bile dis­solved or­ganic car­bon into at­mos­pheric CO₂. Just how much CO₂ they re­lease en masse to the at­mos­phere is yet to be de­ter­mined, but pre­dic­tions are that it will be a lot. Fur­ther­more, SAR11 cells may also con­tribute to the re­lease of methane to the at­mos­phere — a sig­nif­i­cant ac­tiv­ity for cli­mate change since methane is 20-times more po­tent as a green­house gas than CO₂. Ma­rine wa­ters con­tain a sur­pris­ingly high amount of methane. Oceans are 5 − 75% more sat­u­rated with methane than the at­mos­phere, sug­gest­ing that methane pro­duc­tion takes place in ma­rine en­vi­ron­ments and mi­grates from there to the at­mos­phere. Sci­en­tists have for long sought the source of the ocean's methane. They ruled out canon­i­cal methano­gen­e­sis path­ways, as those re­quire strictly anaer­o­bic con­di­tions that are not present in the oceans where methane bio­gen­e­sis is known to oc­cur. To ex­plain the "ma­rine methane para­dox", re­searchers pro­posed that some ma­rine mi­crobes may use the methylphos­pho­nate (Mpn) path­way to scav­enge for phos­phate, thereby pro­duc­ing methane as byprod­uct. Ge­nomic and metage­nomic data pre­dicts that ~10% of bac­te­ria in the oceans are ca­pa­ble of Mpn syn­the­sis, and ~30 − 40% are equipped for Mpn ca­tab­o­lism. Some SAR11 strains (but not P. ubique HTCC1062) are among those that use this path­way when starved for phos­pho­rus and pro­duce methane as a byprod­uct. This, to­gether with their stag­ger­ing abun­dance, sug­gests these bac­te­ria make a sig­nif­i­cant con­tri­bu­tion to the methane emis­sions from the oceans.

P. ubique  se­crets re­vealed by ge­nomics   Tiny as it is, you may not be sur­prised to learn that P. ubique also has a small genome (1,308,759 bp), cur­rently the small­est genome known for a free-liv­ing or­gan­ism. The genome en­codes 1,354 pre­dicted pro­teins, 1 rRNA operon , and 32 tR­NAs. The se­quenced strain (HTCC1062) has no prophages or pseudo­genes, and car­ries an ex­cep­tion­ally low num­ber of par­alogs. Genome data in­di­cate that this bac­terium pos­sesses the com­plete path­ways re­quired to gen­er­ate en­ergy by res­pi­ra­tion and also to har­vest en­ergy from the sun us­ing pro­te­orhodopsin, a light-de­pen­dent, retiny­li­dine pro­ton pump. P. ubique is also equipped with nu­mer­ous high affin­ity nu­tri­ent trans­porters, con­sis­tent with a com­pet­i­tive strat­egy for nu­tri­ent scav­eng­ing. Most are ABC trans­porters, a large and an­cient pro­tein fam­ily noted for pro­vid­ing high sub­strate affini­ties at the cost of co­pi­ous ATP. Clearly, the cells in­vest in trans­port to out­com­pete other mi­crobes in nu­tri­ent ac­qui­si­tion and, based on their abun­dance and ef­fi­cient as­sim­i­la­tion of or­ganic mat­ter, they have suc­ceeded.

Fig­ure 3. Num­ber of pre­dicted pro­tein-cod­ing genes ver­sus genome size for 244 com­plete pub­lished gen­omes from bac­te­ria and ar­chaea. P. ubique has the small­est num­ber of pro­tein-cod­ing genes (1354 open read­ing frames) of any free-liv­ing or­gan­ism. Source

P. ubique en­codes few al­ter­na­tive meta­bolic path­ways in its stream­lined genome. In­stead it has spe­cial­ized as a small, highly ef­fi­cient het­erotroph adapted for com­pet­i­tive suc­cess in an in­vari­able olig­otrophic en­vi­ron­ment. A sin­gle rRNA operon is suf­fi­cient to main­tain their mod­er­ate but steady rate of growth. Fur­ther­more, un­like larger ma­rine het­erotrophs, they do not re­spond by grow­ing faster when pro­vided with more nu­tri­ents. These and other ge­nomic traits sug­gest that P. ubique is un­der strong se­lec­tion pres­sure to keep its genome small. Such a small cell size also con­strains genome size. Even though com­pacted, their chro­mo­some oc­cu­pies about 30% of the cell vol­ume. En­vi­ron­men­tal adap­ta­tion is also sug­gested by the low (29.7%) G+C con­tent of its genome. As one less ni­tro­gen atom is needed in a A‑T bp than in an G‑C bp, ni­tro­gen-lim­ited en­vi­ron­ments such as those in­hab­ited by P. unique of­ten se­lect for low G+C genomes. Fur­ther­more, DNA repli­ca­tion re­quires not only car­bon, ni­tro­gen, and phos­pho­rus as­sim­i­la­tion, but sub­stan­tial amounts of en­ergy. A larger chro­mo­some could com­pro­mise the care­ful bal­ance of meta­bolic ef­fi­ciency and growth. Evo­lu­tion­ary the­ory pre­dicts that se­lec­tion based on the meta­bolic cost of DNA repli­ca­tion will be strongest when the pop­u­la­tion is very large and sta­ble over time. This is pre­cisely the sit­u­a­tion for P. ubique.

Fig­ure 4. Abun­dance of pelagiphage rel­a­tive to repre­sentative cyanophages and roseophage SIO1. Stars de­note pelagiphage genomes iden­ti­fied in this study. To high­light the rel­a­tive abun­dance of HTVC010P com­pared to known cyanophage sub­fam­i­lies, the abun­dance of sub­fam­ily mem­bers iden­ti­fied were summed and are rep­re­sented as clus­ters (ital­ics). Non-ma­rine neg­a­tive con­trol se­quences (En­ter­obac­te­ria phage T4 and fresh­wa­ter Syne­chococ­cus­phage S‑CRM01) were in­cluded in the analy­sis. Source

En­ter the pelagiphages   When the high cell counts for the SAR11 clade were first re­ported, some peo­ple won­dered how these abun­dant 'win­ners' es­caped the kill-the-win­ner (KTW) preda­tor-prey dy­namic. Some pos­tu­lated that they dodged phage pre­da­tion al­to­gether due to their com­bi­na­tion of ex­cep­tion­ally small size, which re­duces the fre­quency of phage-host en­coun­ters, and slow growth, which lim­its the num­ber of prog­eny pro­duced by each suc­cess­ful in­fec­tion. It was also sug­gested that some SAR11 strains might carry prophages that pro­tect them from in­fec­tion by the same or closely re­lated phages. But the fact is that P. ubique car­ries no prophages and it is sub­ject to pre­da­tion by di­verse and abun­dant pelagiphages. The four pelagiphages iso­lated and char­ac­ter­ized to date are all lytic, three of them be­ing stubby-tailed podoviruses (av­er­age genome size of 38,979 bp) and one a my­ovirus (long con­trac­tile tail; 147,284 bp). All four had sim­i­lar la­tent pe­ri­ods (av­er­age 19 − 22 h), but burst size var­ied from 9 (larger my­ovirus) to 43 (smaller podoviruses). The burst sizes may re­flect the num­ber of prog­eny viri­ons that can be made from the re­sources of these small cells — fewer for the larger pelagiphages and more for the smaller.

How does P. ubique stay one step ahead of its phages and main­tain its high abun­dance? It has nei­ther a CRISPR de­fense nor a re­stric­tion-mod­i­fi­ca­tion sys­tem based on DNA methy­la­tion. The an­swer may lie in its strain-level di­ver­sity. KTW acts at the strain level, dec­i­mat­ing phage-sus­cep­ti­ble strains while al­low­ing re­sis­tant ones to pros­per. The net ef­fect is sta­ble num­bers at the species level and a high di­ver­sity of strains. In­deed, genome di­ver­sity in the SAR11 clade is ex­cep­tion­ally high. This was al­ready ap­par­ent from the dif­fi­cul­ties en­coun­tered that pre­vented as­sem­bly of com­plete SAR11 genomes from Sar­gasso Sea datasets de­spite the seem­ingly ad­e­quate depth of cov­er­age. Fur­ther­more, an analy­sis of nine strains of P. ubique iso­lated from a two liter sam­ple of sur­face wa­ter col­lected off the Ore­gon coast re­vealed fre­quent in­traspe­cific re­com­bi­na­tion in seven of the eight genes stud­ied. So high were the mea­sured re­com­bi­na­tion rates that the re­searchers con­cluded: The eu­kary­otic model of species shar­ing a com­mon pool of al­le­les is more apt for this SAR11 pop­u­la­tion than a strictly clonal model of in­her­i­tance in which al­lelic di­ver­sity is con­trolled by pe­ri­odic se­lec­tion. Later work con­firmed and ex­tended this find­ing.

Some of the P. ubique in­ter­strain di­ver­sity also mapped to hy­per­vari­able re­gions in­volved in pri­mary de­fenses against phage in­fec­tion. This in­cluded genes that en­code the cell sur­face com­po­nents that phages re­quire for ad­sorp­tion — their first step en route to in­fec­tion. The arms race be­tween phages and their hosts of­ten plays out in the rapid evo­lu­tion of these host com­po­nents that the phages then counter by mod­i­fi­ca­tions of their host recog­ni­tion and bind­ing pro­teins.

As metage­nomic re­search ac­cel­er­ates, will SAR11 re­tain its ti­tle as the most abun­dant or­gan­ism on the planet? Even if it re­tains its num­ber one rank in ocean wa­ters, glob­ally it might be su­per­seded by one re­sid­ing in the sub­sur­face. The prokary­otes in the sub­sur­face zones be­neath the oceans and the con­ti­nents may be even more abun­dant than those in the ocean wa­ters, as es­ti­mated in Whitman's clas­sic 1998 pa­per. Con­sid­er­ing how lit­tle we know about the mi­cro­bial com­mu­ni­ties from those less ac­ces­si­ble realms, it seems pos­si­ble that a new con­tender will be dis­cov­ered and SAR11 will be de­throned. Would any­one care to make a wa­ger?

 

Ref­er­ences

• Gio­van­noni SJ, Tripp HJ, Gi­van S, Po­dar M, VerginKL, Bap­tis­taD, Bibbs L, Eads J, Richard­son TH, No­ordewier M. 2005. Genome stream­lin­ing in a cos­mopoli­tan oceanic bac­terium. Sci­ence, 309, 1242−1245. PMID 9618454
• Karl DM, Bev­ers­dorf L, Björk­man KM, Church MJ, Mar­tinez A, De­long EF. 2008. Aer­o­bic pro­duc­tion of methane in the sea. Na­ture Geo­science, 1,473−478. DOI 10.1038/ngeo234
• Mor­ris RM, Rappé MS, Con­non SA, Ver­gin KL, Siebold WA, Carl­son CA, Gio­van­noni SJ. 2002. SAR11 clade dom­i­nates ocean sur­face bac­te­ri­o­plank­ton com­mu­ni­ties. Na­ture, 420, 806−810. PMID 12490947
• Zhao, Y, Tem­per­ton B, Thrash JC, Schwal­bach MS, Ver­gin KL, Landry ZC, El­lis­man M, Deer­inck T, Sul­li­van MB, Gio­van­noni SJ. 2013. Abun­dant SAR11 viruses in the ocean. Na­ture, 494, 357−360. PMID 23407494

 

Merry Youle is a pha­geophilic mi­cro­bi­ol­ogy writer/editor liv­ing on the Big Is­land of Hawaii and STC blog­ger emerita.
Gemma Reguera is as­so­ciate pro­fes­sor in the De­part­ment of Mi­cro­bi­ol­ogy and Mol­e­c­u­lar Ge­net­ics, Michi­gan State Uni­ver­sity and an As­so­ciate Blog­ger at STC.