The Un­cul­tured Bac­te­ria

by Kim Lewis

Fig­ure 1. "The Great Plate Count Anom­aly."

The ma­jor­ity of bac­te­ria will not grow on nu­tri­ent medium in the lab. The ba­sic ex­per­i­ment is sim­ple: take a sam­ple from the en­vi­ron­ment, such as ma­rine sed­i­ment or soil, mix with wa­ter, vor­tex, al­low it to set­tle, di­lute su­per­natant and take two droplets. Plate one on a Petri dish with LB medium, and place the other on a mi­cro­scope slide (adding a dye such as DAPI helps). Count the num­ber of cells un­der the mi­cro­scope and the num­ber of colonies on the Petri dish — the re­sult will be "The Great Plate Count Anom­aly." About 100 times more cells will be ob­served mi­cro­scop­i­cally than colonies counted on the Petri dish (Fig. 1). This sim­ple re­sult is one of the most pro­found puz­zles in mi­cro­bi­ol­ogy, and one of the most sig­nif­i­cant un­solved ques­tions in bi­ol­ogy. In­deed, mi­croor­gan­isms prob­a­bly make up the bulk of the to­tal bio­di­ver­sity of species on the planet, but we do not have ac­cess to the vast ma­jor­ity of them. Im­por­tantly, most Di­vi­sions — the largest tax­o­nomic units — do not have a sin­gle cul­tivable rep­re­sen­ta­tive, and we know of their ex­is­tence only from 16S rDNA iso­lated di­rectly from the en­vi­ron­ment.

Fig­ure 2. Grow­ing the un­cul­tured in agar, be­tween semi-per­me­able mem­branes, in their orig­i­nal en­vi­ron­ment.

There are two ba­sic ap­proaches to solv­ing a prob­lem, and one of them is to de­cide that it does not ex­ist. It has been sug­gested on the pages of Na­ture and Mi­crobe that the anom­aly only ex­ists in our minds and re­sults from the in­suf­fi­cient num­ber of mi­cro­bi­ol­o­gists with a "green thumb" will­ing to tin­ker with growth con­di­tions. How­ever, the anom­aly has ex­isted for over 100 years, and tin­ker­ing by gen­er­a­tions of sci­en­tists pro­duced only mi­nor im­prove­ments. A se­ri­ous ef­fort was mounted by sev­eral groups to cul­ture rep­re­sen­ta­tives from the ubiq­ui­tous TM7 di­vi­sion, for ex­am­ple, but pro­duced no re­sults.

What I find most fas­ci­nat­ing in the prob­lem of un­cul­tured bac­te­ria, how­ever, is not the ex­is­tence of ex­otic strangers such as TM7, but the fact that many — if not most — "un­cul­tur­able" or­gan­isms are close rel­a­tives of com­mon gar­den-va­ri­ety bac­te­ria such as Bacil­lus sub­tilis or Pseudomonas aerug­i­nosa. Judg­ing by the genomes of their rel­a­tives, the un­cul­tured or­gan­isms should grow on al­most any­thing, yet they grow on noth­ing in the lab, in­clud­ing yeast ex­tract which con­tains the en­tire meta­bolic map. They do grow in soil or ma­rine sed­i­ment, so should be able to con­sume degra­da­tion prod­ucts of plants and an­i­mals, which would be sug­ars and amino acids. The puz­zle is truly per­plex­ing.

Fig­ure 3. A sam­ple from ma­rine sed­i­ment biofilm is in­oc­u­lated on a Petri dish with rich medium (left panel). In­oc­u­lat­ing colonies from this plate pair­wise (right panel) al­lows to iden­tify un­cul­tured or­gan­isms and their helpers. Ma­te­r­ial from one colony, KLE1104 (close rel­a­tive of cul­tivable M. poly­si­pho­niae) was spread evenly over the plate, while cells from an­other colony, KLE1011 (re­lated to M. lu­teus) were patched in a sin­gle spot. KLE1104 colonies only form around KLE1011.

Given the un­suc­cess­ful 100 year-old quest for a good medium, we gave up on the hope of recre­at­ing it in a Petri dish. In­stead, we de­cided to grow the un­cul­tured in their nat­ural en­vi­ron­ment, where one can­not pos­si­bly fail! En­clos­ing a ma­rine sed­i­ment sam­ple mixed with agar be­tween semi-per­me­able mem­branes and plac­ing it back in its orig­i­nal en­vi­ron­ment then al­lows for free dif­fu­sion of com­pounds through the cham­ber (Fig. 2). The bac­te­ria are tricked into per­ceiv­ing this dif­fu­sion cham­ber as their nat­ural habi­tat, and form colonies. Sim­i­lar ap­proaches of cul­tur­ing in their nat­ural en­vi­ron­ment re­sulted in cul­ti­va­tion of dom­i­nant pelagic Pelag­ibac­ter ubique by Steve Giovannoni's group, and Prochloro­coc­cus by Zinser and co-au­thors.

One use­ful clue to the na­ture of un­cul­tivabil­ity came from our ob­ser­va­tion that some un­cul­tured or­gan­isms will only grow in the pres­ence of cul­tivable species from the same en­vi­ron­ment (Fig. 3). Us­ing this as an as­say, in col­lab­o­ra­tion with Jon Clardy's group we dis­cov­ered that growth fac­tors for many un­cul­tured species from biofilms en­velop­ing ma­rine sand grains are siderophores, chela­tors of in­sol­u­ble Fe(III) (Fig. 4). Some species are fairly promis­cu­ous in tak­ing up siderophores, while oth­ers, such as a dis­tant rel­a­tive of Ver­ru­comi­cro­bia, will only grow in the pres­ence of a siderophore from a par­tic­u­lar neigh­bor.

Fig­ure 4. A cul­tur­able helper re­leases a siderophore that cap­tures in­sol­u­ble Fe(III) and brings it into the cell. The same siderophore/ Fe(III) com­plex is cap­tured by an un­cul­tured bac­terium which is un­able to make its own siderophores.

But why would bac­te­ria lose the abil­ity to make a fac­tor nec­es­sary for cap­tur­ing an es­sen­tial nu­tri­ent, thus be­com­ing de­pen­dent on their neigh­bors? This de­pen­dency, of course, leads to loss of lib­erty; un­cul­tured species can no longer col­o­nize new ter­ri­tory. Sim­ple ad­van­tages of thiev­ery are un­likely to pro­vide a cred­i­ble ex­pla­na­tion. In­deed, siderophore piracy is well-known among bac­te­ria, but the cul­tivable pi­rates re­tain their own siderophore oper­ons and turn ex­pres­sion on when iron gets low. I think that the rea­son for the loss of siderophores is to pre­vent adap­tive evo­lu­tion in new en­vi­ron­ments. Any time a cell finds it­self in new sur­round­ings, it will evolve to in­crease its fit­ness. This newly evolved or­gan­ism is un­likely to out­com­pete res­i­dent species that spent mil­lions of years adapt­ing to the same en­vi­ron­ment, and will die out. Go­ing back to their orig­i­nal en­vi­ron­ment is not an op­tion ei­ther; they are not the same now and will be less fit than their par­ents (Fig. 5).

Fig­ure 5. Up­per panel Death by an evo­lu­tion­ary dead-end. A cul­tur­able bac­terium prop­a­gates in its fa­mil­iar nich (yel­low) and sheds off a cell that trav­els to a new en­vi­ron­ment (grey) where it at­tempts to adapt by evolv­ing new fea­tures and los­ing some old ones. It is un­able how­ever to com­pete with a res­i­dent species (pur­ple), and is no longer com­pet­i­tive with the par­ent strain. Lower panel An un­cul­tured or­gan­ism de­pends on a growth fac­tor from a neigh­bor­ing species. If a cell finds it­self in an un­fa­mil­iar en­vi­ron­ment, it stops di­vid­ing and waits for a ride back, where it re­sumes pro­duc­tive prop­a­ga­tion.

This sce­nario has been de­scribed for our pathogens such as P. aerug­i­nosa which looses a large num­ber of genes, in­clud­ing vir­u­lence fac­tors and pro­teases, while adapt­ing to the en­vi­ron­ment of the lung of cys­tic fi­bro­sis pa­tients. The re­sult of such run­away evo­lu­tion is a dead end; the pathogen can nei­ther in­fect new hosts nor re­turn to soil, its other ma­jor habi­tat. This is prob­a­bly the main fate of weeds like P. aerug­i­nosa or E. coli — death by adap­tive evo­lu­tion. The other 99% of species choose where they live and grow. If they find them­selves in an un­fa­mil­iar en­vi­ron­ment, then the best strat­egy is to go into dor­mancy and wait for a ride back home. This strat­egy is very sim­i­lar to what we know about spores of cul­tivable species such as B. sub­tilis. Spores will ger­mi­nate on any nu­tri­ent en­vi­ron­ment, but the prob­a­bil­ity of ger­mi­na­tion is greatly in­creased in the "cor­rect" en­vi­ron­ment. Ala­nine (for rea­sons we do not un­der­stand) is a good ger­mi­na­tor, and so are prod­ucts of pep­ti­do­gly­can hy­drol­y­sis from neigh­bor­ing species. B. sub­tilis seems to be an in­ter­me­di­ate be­tween a true un­cul­tured species and a weed like E. coli.

What is next? The siderophore story sug­gests that there are other growth fac­tors to be dis­cov­ered that in­di­cate a fa­mil­iar en­vi­ron­ment to the un­ad­ven­tur­ous un­cul­tured. (Next year: the un­cul­tured from the Hu­man Mi­cro­biome.)

Ref­er­ence

D'Onofrio A, Craw­ford JM, Stew­art EJ, Witt K, Gavr­ish E, Ep­stein S, Clardy J, Lewis K. (2010). Siderophores from neigh­bor­ing or­gan­isms pro­mote the growth of un­cul­tured bac­te­ria. Chem­istry & bi­ol­ogy, 17 (3), 254−264. PMID 20338517

All fig­ures cour­tesy of Kim Lewis.

Kim Lewis is Pro­fes­sor of Bi­ol­ogy and Di­rec­tor of the An­timi­cro­bial Dis­cov­ery Cen­ter at North­east­ern Uni­ver­sity.