The Bac­te­r­ial Con­sor­tium Chlorochro­matium ag­gre­ga­tum

by Mark O. Mar­tin

With apolo­gies to the old Turtle's song (which knocked the Beatle's "Penny Lane" off the Bill­board charts in the spring of 1967), there are many ways that mi­crobes in­teract with one an­other. Some in­ter­mi­cro­bial in­ter­ac­tions can be neg­a­tive, rang­ing from com­pe­ti­tion for re­sources to pre­da­tion. Oth­ers can be es­sen­tially in­dif­fer­ent to­ward one mem­ber, as in com­men­sal­ism. Some of the most in­teresting as­so­ci­a­tions, though, are the ones in which both mem­bers ben­e­fit: mu­tu­al­ism. Be­ing an en­thu­si­as­tic fan of the mi­cro­bi­o­log­i­cally off­beat, I have been de­lighted in re­cent years to see how mod­ern tools (and some very hard work) have be­gun to re­veal the de­tails of mi­crobe-mi­crobe in­ter­ac­tions within each of these cat­e­gories.

Fig. 1.C. ag­gre­ga­tum ma­k­es the cover. Source

Re­cently, Small Things Con­sid­ered posted a fas­ci­nat­ing es­say de­scrib­ing progress in the study of an as­so­ci­a­tion be­tween two ob­lig­ately in­ter­re­lated ar­chaea, Nanoar­ch­aeum eq­ui­tans and Ig­nic­oc­cus hos­pi­talis. Mi­crobe-mi­crobe com­men­sal­ism or mu­tu­al­ism is not un­heard of (there are also some fas­ci­nat­ing ex­am­ples in this blog), but are only now be­ing in­tensively in­vestigated. Con­sor­tia are as­sem­blages of dif­fer­ent species of mi­crobes in phys­i­cal – and some­times in­tri­cate bio­chem­i­cal – con­tact with one an­other, and are im­pli­cated in bi­o­log­i­cal processes rang­ing from sewage treat­ment to ma­rine ni­tro­gen cy­cling to meta­bolic processes within the ru­men. In my opin­ion, they re­flect the "real world" of mi­cro­bi­ol­ogy far more than sin­gle colony iso­lates on Petri plates or in broth cul­tures!

Fig. 2. A trans­verse sec­tion of Chloro­chro­matium. The cen­tral cell is the motile an­aerobic het­erotroph, the seven at­tached to it, the green sul­fur bac­te­ria. Bar = 0.5 μm. Source

This brings us to the tan­ta­liz­ing story of Chlorochroma­tium ag­gre­ga­tum, a con­sor­tium that has been in­ten­sively stud­ied by Jörg Over­mann and his cowork­ers for a num­ber of years (here is a very in­for­ma­tive link to his labora­tory web­site at Lud­wig-Max­i­m­il­ians-Uni­ver­sität, Mu­nich. in­ac­tive as of 2021) I am par­tic­u­larly im­pressed by this group's dis­ci­pli­nary con­flu­ence of ul­tra­struc­tural EM analy­sis, mol­e­c­u­lar bi­ol­ogy, bio­chem­istry, bioin­for­mat­ics, bio­geog­ra­phy, and mi­cro­bial ecol­ogy.

This in­trigu­ing con­sor­tium, of­ten found in the chemo­cline of strat­i­fied fresh­wa­ter lakes world­wide, is well adapted to take ad­van­tage of low light and low sul­fide anaer­o­bic con­di­tions. It con­sists of a motile chemo­heterotrophic rod and a num­ber of green sul­fur bac­te­r­ial epibionts. The mem­bers of the as­so­ci­a­tion have been stud­ied by use of en­rich­ment cul­ture, mi­cro­ma­nip­u­la­tion, and PCR-based 16S rRNA cen­suses. (By the way, I try to be care­ful about us­ing the word metage­nomics when only 16S rRNA se­quences are ob­tained and an­a­lyzed. Al­though the "16S rRNA cen­suses" is a bit pon­der­ous, it is also more accu­rate.) Be­tween 20 and 70 epibionts cover the sur­face of the cen­tral rod, as seen in Fig­ure 1. The cen­tral rod with a sin­gle po­lar fla­gel­lum is a β‑proteobacter within fam­ily Co­ma­mon­adaceae (genus level rel­a­tives in­clude Var­i­ovo­rax, Rhod­oferax, and Delf­tia). The green sul­fur bac­te­r­ial epibionts are re­lated to more "typ­i­cal" green sul­fur bac­te­ria, com­plete with chloro­somes and an au­totrophic re­verse TCA path­way to as­sim­i­late car­bon. At least seven dif­fer­ent types of motile pho­totropic con­sor­tia of this type have been found to date. They make up two-thirds of the bio­mass found in the chemo­cline of fresh­wa­ter lakes. The Over­mann group has also shown that at least nine­teen dif­fer­ent phy­lo­types of green sul­fur bac­te­r­ial epibionts are found in this kind of as­so­ci­a­tion world­wide; the epibionts do not ap­pear to be mono­phyletic, sug­gest­ing that such con­sor­tia have evolved in­de­pen­dently sev­eral times, or that the ca­pac­ity to form them was pre­sent at the be­gin­ning of the green sul­fur bac­te­r­ial clade ra­di­a­tion.

Fig. 3. Sco­to­pho­bic re­sponse of C. aggre­gatum. Phase- con­trast pho­tomi­cro­graph at the end of the accu­mulation pe­riod. Up­per = closed field stop. Lower = open field stop. Bar = 50 mm. Source

There are three lines of ev­i­dence that the epibionts and the motile cen­tral rod are in some form of rapid and com­plex com­mu­ni­ca­tion. First, the con­sor­tium dis­plays sco­to­phob­o­taxis; as the cells leave light and en­ter dark­ness, they re­verse di­rec­tion back into the light. Since the epibionts are non­motile, the cen­tral rod (which posses­ses no pho­to­syn­thetic mem­branes) re­sponds to light via some sort of bio­chem­i­cal com­mu­ni­ca­tion with its pho­to­synthetic part­ner. This prop­erty can be ob­served un­der a mi­cro­scope (Fig­ure 3), and has been used to en­rich envi­ronmental sam­ples for C. ag­gre­ga­tum. Sec­ond, microau­toradiography has demon­strated that al­pha-ke­tog­lu­tar­ate is rapidly taken up by the cen­tral rod, but only in the pres­ence of light and sul­fide-stim­u­lated epibiont ac­tiv­ity. Fi­nally, there ap­pears to be tight syn­chro­niza­tion be­tween di­vi­sion rates of the cen­tral rod and the epibionts.

The Over­mann lab used 16S rRNA "mark­ers" spe­cific to each mem­ber of the con­sor­tium. Us­ing FISH analy­sis, they were able to show that nei­ther part­ner is found by it­self in nat­ural pop­u­la­tions. How­ever, the re­searchers were able to cul­ti­vate the green sul­fur bac­te­r­ial epibiont in pure cul­ture (prov­ing that it was not ob­lig­ately as­so­ci­ated with the cen­tral rod). This or­gan­ism, named Chloro­bium chlorochro­matii, was shown to be sim­i­lar to other green sul­fur bac­te­ria: it is ob­lig­ately anaer­o­bic, pos­sesses chloro­somes, uses sul­fide as an elec­tron donor, and so forth.

This group re­cently pub­lished a pa­per about spe­cific ul­tra­struc­tural as­pects of the con­sor­tium. Us­ing high-res­o­lu­tion cryosub­sti­tuted SEM, TEM, and care­ful im­age re­con­struc­tion, they ob­served struc­tures that may be spe­cific to the as­so­ci­a­tion. Fig­ure 4 shows a di­a­gram out­lin­ing a "geo­gra­phy" of this con­sor­tium, in­clud­ing mys­te­ri­ous struc­tures within and be­tween both epibiont and cen­tral rod.

Fig. 4. Schematic rep­re­sen­ta­tion from 3D recon­struction of Chlorochro­matium ag­gregatum. Epibionts at­tach by long, hair-like poly­sac­cha­ride chains. LB = lipid bo­dies with a myelin-like pat­tern at­tached to the cy­to­plas­mic mem­brane. PP = polyphos­phate glob­ules. The at­tach­ment site of the epibiont is char­ac­ter­ized by the ab­sence of chloro­somes and a sin­gle con­tact layer, the ECL. Strik­ing fea­tures of the fla­gel­lated cen­tral bac­terium are (i) periplas­mic tubules (PT) that can be in di­rect con­tact with the epibionts, postu­lated to form a com­mon periplas­mic space; (ii) com­plex in­vagi­na­tions of the cy­to­plas­mic mem­brane (MW = membran­ous whirls); (iii) sub­units arranged in small mono­lay­ers or bi­lay­ers di­rectly as­so­ciated with the cy­to­plas­mic mem­brane (CML); and (iv) paracrys­talline struc­tures (CBC) on the in­ner side of the cyto­plasmic mem­brane (or mem­bra­nous in­va­ginations). Source

Both mem­bers of the con­sor­tium have un­usual ultra­structural com­po­nents. The epibionts pos­sess a rough sur­face with many pro­tru­sions; the vol­ume of the pe­riplasm varies from lo­ca­tion to lo­ca­tion. There are also var­i­ous in­tra­cel­lu­lar glob­ules that ap­pear to be lipid bod­ies and polyphos­phate in­clu­sions. The cen­tral rod has sur­face vari­a­tions and papil­lae-like ex­ten­sions of the outer mem­brane (see be­low). These cells also con­tain in­tra­cel­lu­lar "zip­per-like" reg­u­lar struc­tures (cen­tral bac­te­r­ial crys­tals or CBCs), as well as "mem­bra­nous whirls" con­sist­ing of ag­gre­gates of cy­to­plas­mic tubule-like struc­tures.

Sev­eral ul­tra­struc­tural prop­er­ties may be rel­e­vant to the as­so­ci­a­tion of mem­bers of the con­sor­tium. First, a re­markable se­ries of fine fil­a­ments cover the epibiont sur­faces, ap­pear­ing some­thing like an "elas­tic net" that holds the en­tire con­sor­tium to­gether. It is tempt­ing to con­sider these fil­a­ments as com­posed of poly­sac­cha­ride-like ad­hesins, but their na­ture is still un­der in­ves­ti­ga­tion. Sec­ond, the re­gion where the epibionts "touch" the cen­tral rod – the epibiont con­tact layer (ECL) – has a unique pla­nar ap­pear­ance. The lack of chloro­somes in the ECL may fa­cil­i­tate metabo­lite trans­fer be­tween the two com­ponent or­gan­isms. Fi­nally, there may be a com­mon pe­riplasm be­tween the epibionts and the cen­tral cell. This ex­cit­ing ob­ser­va­tion sug­gests pos­si­ble di­rect cell-cell com­mu­ni­ca­tion within the con­sor­tium.

Re­cently, Over­mann and col­leagues ap­plied sup­pres­sion-sub­trac­tive hy­bridiza­tion to what is known of the genomes of 16 free-liv­ing green sul­fur bac­te­r­ial to search for genes spe­cific to this mu­tu­al­is­tic as­so­ci­a­tion. Can­di­dates so far in­clude genes with ad­hesin-like prop­er­ties and one with a cal­cium-bind­ing mo­tif – pos­si­bly sig­nif­i­cant, as sub­se­quent ex­per­i­ments showed that cal­cium chela­tors dis­rupt the con­sor­tium.

Clearly, there is a great deal of hard work ahead and in­ter­est­ing re­sults to be re­vealed. I know I will be watch­ing the jour­nals to see what re­sults ap­pear next re­gard­ing this "happy as­so­ci­a­tion" be­tween a motile rod and its pho­to­syn­thetic part­ner. Just how does this con­sor­tium adapt suc­cessfully to liv­ing deep within lakes, with no oxy­gen, lit­tle sul­fide, and dim light? Dim though their world may be, I sus­pect that this con­sor­tium will shed a great deal of light on top­ics rang­ing from sym­bioses to in­ter­cel­lu­lar com­mu­ni­ca­tion to ecol­ogy to mul­ti­cel­lu­lar as­so­ci­a­tions. Happy to­gether...

 

Mark is as­so­ciate pro­fes­sor in the De­part­ment of Bi­ol­ogy, Uni­ver­sity of Puget Sound, and a pas­sion­ate ad­vo­cate for the Small Things..