The Three Faces of Thiomar­garita

Bud­ding is ev­i­dent on these at­tached, elon­gated Thiomar­garita-like bac­te­ria col­lected near a methane seep off the coast of Costa Rica. Bar = ~1 mm. Source

by Merry Youle

How in­ap­pro­pri­ate to call this planet Earth when it is quite clearly Ocean.
—Arthur C. Clarke

Ahhh, the de­li­cious aroma of hy­dro­gen sul­fide. When as kids we en­coun­tered  it, we would hold our nose and pro­claim "yuck, rot­ten eggs!" It is in­deed pro­duced by some­thing 'rot­ting,' but specif­i­cally rot­ting un­der anaer­o­bic con­di­tions, such as in swamps and sew­ers. It is also abun­dant in coastal ma­rine sed­i­ments, pro­duced by the oxy­gen-con­sum­ing het­erotrophic de­com­po­si­tion of or­ganic mat­ter. This makes for a layer at the sea bot­tom that is sul­fide rich but oxy­gen poor. Nev­er­the­less, leav­ing no po­ten­tial en­ergy source un­tapped, some bac­te­ria in these zones use sul­fur ox­i­da­tion as their en­ergy source. They in­clude two re­mark­ably large γ‑proteobacteria, Thio­ploca and Thiomar­garita. Thio­ploca copes with the lack of oxy­gen by us­ing ni­trate as an al­ter­nate ter­mi­nal elec­tron ac­cep­tor, but this just ex­changes one prob­lem for an­other. The sul­fides are in the sed­i­ment, the ni­trate in the wa­ter col­umn above. So Thio­ploca com­mutes be­tween the two zones, as Elio de­scribed in an ear­lier post.

Thiomar­garita nami­bi­en­sis, the "Namib­ian sul­fur pearl." Cour­tesy of the Mi­cro­bi­o­log­i­cal Gar­den. Source

Non-motile Thiomar­garita uses a dif­fer­ent tac­tic. This bac­terium was first dis­cov­ered in 1999 off the Namib­ian coast, thus was named T. nami­bi­en­sis. Its cells are large spheres, arranged in chains, each chain en­closed in a mu­cous sheath. Av­er­age cell di­am­e­ter is 180 μm, with rare in­di­vid­u­als reach­ing 750 μm, which is about as big as bac­te­r­ial cells get. But they do this by cheat­ing. Most of the 'cell' is a large vac­uole, with the cy­to­plasm rel­e­gated to a thin sur­round­ing outer layer. Sul­fur glob­ules are ev­i­dent in the cy­to­plasm. Thus the genus name, Thiomar­garita, that was de­rived from the Greek for 'sul­fur pearl.' Thiomar­garita spp. are wide­spread in hy­dro­gen sul­fide-rich coastal sed­i­ments. They, too, use ni­trate as their ter­mi­nal elec­tron ac­cep­tor. Their strat­egy is to take ad­van­tage of the oc­ca­sional events that re­sus­pend the sed­i­ment and bring abun­dant ni­trates into the wa­ter col­umn. They then hoard ni­trate in their huge vacuoles—enough ni­trate to see them through even months of scarcity.

Thiomar­garita from seeps in the Gulf of Mex­ico re­pro­duce by re­duc­tive di­vi­sion in three di­men­sions, form­ing clus­ters of 2, 4, 8, or 16 cells. Source

But not all Thiomar­garita form the char­ac­ter­is­tic "chain of pearls." Ex­plo­ration of hy­dro­car­bon seeps in the Gulf of Mex­ico in 2005 turned up Thiomar­garita-like cells with a dis­tinctly dif­fer­ent mor­phol­ogy. These or­gan­isms un­dergo a re­duc­tive di­vi­sion to yield clus­ters of 2, 4, 8, or 16 cells, each with the char­ac­ter­is­tic Thiomar­garita-like spher­i­cal mor­phol­ogy with a large vac­uole and a thin layer of cy­to­plasm with sul­fur glob­ules.

Yet a third mor­pho­log­i­cal va­ri­ety has been re­cently dis­cov­ered. Re­searchers ex­plor­ing the ecol­ogy of methane seeps along the Pa­cific mar­gin of Costa Rica (depths of 990‑1600 m) rec­og­nized the fa­mil­iar Thiomar­garita forms in the sed­i­ment. But in some re­gions they also no­ticed "thin films of mi­cro­bial bio­mass" cov­er­ing many sur­faces.

A close look re­vealed what ap­pears to be a dense car­pet of elon­gated Thiomar­garita-like bac­te­ria. These thickly cover some of the methane-de­rived au­thi­genic car­bon­ate rocks and the byssal threads that at­tach cer­tain mus­sels to the sub­strate. Most fre­quently they're seen ad­her­ing to var­i­ous in­ver­te­brates. And quite an amaz­ing sight they make! The at­tached cells have that Thiomar­garita look: a thin-layer of sul­fur glob­ule-con­tain­ing cy­to­plasm sur­round­ing a sin­gle large vac­uole. They're av­er­age length is 338 μm, but some stretch to 700 μm. Small, spher­i­cal cells of­ten ap­pear to be bud­ding from their un­at­tached poles. The re­searchers brought sam­ples on board so they could fol­low this bud­ding process for their en­tire ten day cruise. From this they cal­cu­lated that each day one bud in 300 de­tached from the par­ent cell.

Elon­gate Thiomar­garita-like cells at­tached to Ba­thy­modi­o­lus mus­sel byssal threads. Bar = 500 μm. Source

Putting two and two to­gether, one can well imag­ine that these Thiomar­garita have a di­mor­phic life cy­cle. Sup­pose the elon­gated at­tached cells re­pro­duce by bud­ding, and the freed cells later at­tach to a sub­strate and elon­gate, thus com­plet­ing the cy­cle. If so, then the at­tached elon­gated cells and at least some of the free-liv­ing spher­i­cal cells are sim­ply dif­fer­ent stages in the life cy­cle of the same species. Some­thing like a gi­ant Caulobac­ter! Al­though the ev­i­dence is not yet wa­ter­tight, this hy­poth­e­sis seems likely to me. These re­searchers not only ob­served the bud­ding process, but also saw that the at­tached cells spanned the en­tire spec­trum of ex­pected shapes from nearly spher­i­cal, re­cently-landed cells to slightly elon­gated to fully elon­gated and bud­ding. Lots of ques­tions re­main. So far, there is no way to es­ti­mate what pro­por­tion of the soli­tary cells arose by bud­ding or what por­tion of the gen­er­a­tion time is spent be­ing plank­tonic ver­sus at­tached.

Bud­ding caught in the act. The ar­row 3 in­di­cates ini­tial bud for­ma­tion, ar­row 5 a spher­i­cal daugh­ter cell. Bar = ~350 μm. Source

If the plank­tonic and at­tached forms are the same species, their 16S rRNA gene se­quences would be iden­ti­cal. To test this, the re­searchers iso­lated in­di­vid­ual cells of var­i­ous shapes. They then PCRed the 16S rRNA genes in these sin­gle cells us­ing primers de­signed for Thiomar­garita-re­lated or­gan­isms. When they se­quenced the PCR prod­ucts, they found that one plank­tonic cell and one at­tached cell had es­sen­tially iden­ti­cal 16S se­quences. (COS008 and COS014 shared 99.4% iden­tity, which is within the mar­gin of er­ror.) When they built a phy­lo­ge­netic tree that also in­cluded other Thiomar­garita spp., they found that the var­i­ous Thiomar­garita-like cells col­lected from Costa Rica were placed in at least three dis­tinct clus­ters. Most of them are re­lated to pre­vi­ously-known Thiomar­garita spp., but two (COS001 and COS016) sit on their own sep­a­rate branches.

Phy­lo­ge­netic tree of 16S rRNA gene se­quences of sul­fide-ox­i­diz­ing γ‑proteobacteria. Grey = se­quences of dif­fer­ent mor­pho­types from Costa Rica (COS) and Namibia (NAM). For each cell se­quenced in this study, the mor­phol­ogy and di­am­e­ter are in­cluded. Bar = ~10% se­quence di­ver­gence. Source

These same at­tached forms have also been ob­served at methane seeps off the coast of Ore­gon. Why are elon­gated Thiomar­garita-like cells found around these seeps but not, so far, else­where? The au­thors sug­gest that elon­ga­tion may be a strat­egy to en­hance phos­phate up­take, as had been pre­vi­ously posited for the α‑proteobacterium Caulobac­ter cres­cen­tus. This hy­poth­e­sis as­sumes that in or­der to main­tain a cer­tain amount of cy­to­plasm, Thiomar­garita needs both a large enough sur­face area for  phos­phate up­take and a large enough vac­uole for ni­trate stor­age. One way to have both is by elon­gat­ing. An­other way to achieve the same result—larger sur­face area for the same ni­trate stor­age capacity—is by pro­duc­ing smaller cells, the op­tion used by the re­duc­tively-di­vid­ing clus­ters.

And here the story rests. An­swer­ing the pend­ing ques­tions won't be easy, as the or­gan­isms can­not be cul­tured and col­lect­ing sam­ples re­quires a deep-ocean re­search sub­mersible. How­ever, the bot­tom of the sea is a never-end­ing source of sur­prises and ex­plor­ing the deeps is guar­an­teed to yield yet more amaz­ing new find­ings.

Ref­er­ence

Bai­ley JV, Salman V, Rouse GW, Schulz-Vogt HN, Levin LA, & Or­phan VJ (2011). Di­mor­phism in methane seep-dwelling eco­types of the largest known bac­te­ria. The ISME Jour­nal, 5 (12), 1926–35 PMID: 21697959