Are You Me or Am I You?

by Elio

When one cell meets an­other, it must make de­ci­sions. This ex­tends it­self to prac­ti­cally every at­tribute of liv­ing things, from avoid­ing mat­ing with self to the es­tab­lish­ment of ter­ri­to­r­ial bound­aries. Do we fuse? Will you at­tack me? Are you a po­ten­tial mate or are you lunch? And, in the bod­ies of ver­te­brates, are you some­thing that my im­mune sys­tem should rec­og­nize as for­eign? Mi­crobes are not ex­empted from these quan­daries, be­ing that they carry out lively con­ver­sa­tions for pur­poses as di­verse as feed­ing, dif­fer­en­ti­at­ing into biofilms or fruit­ing bod­ies, mov­ing, sur­viv­ing in a host, or sens­ing their own num­bers. As with the so­cial in­sects, mi­crobes con­verse in a chem­i­cal lan­guage. As Strass­man et al. say in an in­sight­ful re­view: Recog­ni­tion seems likely to be par­tic­u­larly im­por­tant to mi­crobes be­cause they un­der­take many processes ex­tra­cel­lu­larly in the pub­lic sphere that larger or­gan­isms pri­va­tize in­side.

Growth of Pro­teus on an agar plate, start­ing from an in­ocu­lum in the cen­ter. Source

In study­ing such mi­cro­bial in­ter­ac­tions, one can choose among a num­ber of in­ter­est­ing mod­els. The Gram-neg­a­tive bacil­lus, Pro­teus mirabilis, prac­ti­cally begs us to se­lect it.

This com­mon cause of re­cur­rent uri­nary tract in­fec­tions has been known for ages for its some­what cu­ri­ous be­hav­ior on agar plates. Un­like most bac­te­r­ial colonies one is likely to en­counter, which de­velop within nar­row con­fines, those of P. mirabilis spread over the sur­face by a be­hav­ior called swarming—in it­self an in­ter­est­ing ex­am­ple of mi­cro­bial dif­fer­en­ti­a­tion. Con­sider an agar plate in the cen­ter of which a sin­gle P. mirabilis cell has been in­oc­u­lated. The cell will grow and di­vide, like any well-be­haved bac­terium. But af­ter a while, the re­sult­ing cells be­come elon­gated, sprout ex­tra fla­gella, and start mov­ing rapidly out­wards. These so-called swarmer cells ex­tend over the sur­face of the agar and, one as­sumes, over the sur­face of the ep­ithe­lium of the hu­man uri­nary tract. While they do this, they don't grow much, they just move a lot. At some point, these long swarmer cells stop mov­ing and those at the edge of the swarm di­vide into shorter cells. These 'nor­mal sized' cells grow for a while be­fore they dif­fer­en­ti­ate again into swarm­ers, leav­ing be­hind a vis­i­ble ring of cells on the agar as they re­peat the process. The re­sult is that what you see on the plate is a se­ries of con­cen­tric growth rings, mak­ing it look like a bulls­eye. (For a video that shows P. mirabilis cells swarm­ing on an agar plate, click here.) Not much is known about the sig­nals that lead to the dif­fer­en­ti­a­tion of small grow­ing cells and long swarmer cells. Swarmingis a bane of clin­i­cal mi­cro­bi­ol­o­gists who try to pick colonies from a patient's cul­tured sam­ple con­tain­ing many kinds of bac­te­ria, since Pro­teus will over­grow the oth­ers.

Pro­teus mirabilis ter­ri­to­r­ial bound­aries in a swarm plate of the wild-type strain BB2000, BB2000 idsABCDEF dele­tion mu­tant (Δids), BB2000 with the idsA-gfp vec­tor pKG100 (la­beled pPr-ids-gfp), or with an empty vec­tor. A vis­i­ble bound­ary formed be­tween swarms of the wild type and the dele­tion mu­tant. Bar = 1 cm. Source

Also known for a long time is that if you start two colonies of P. mirabilis on a sin­gle plate, you can get two dif­fer­ent out­comes de­pend­ing on their iden­tity. If both colonies are from the same clone, the swarms will sim­ply make con­tact and melt to­gether. Swarms from dif­fer­ent strains, on the other hand, make a vis­i­ble bound­ary in the form of a cell-free zone. This has been known as the Di­enes line (af­ter Louis Di­enes, who de­scribed the phe­nom­e­non in 1946), and is used to dis­tin­guish clin­i­cal iso­lates. What we have here is recog­ni­tion of self and non-self. The mech­a­nism in­volved re­mains some­what murky and lit­tle progress has been made in iden­ti­fy­ing pos­si­ble se­creted mol­e­cules. But there is much hope be­cause this topic is be­ing stud­ied in earnest.

In 2008, Gibbs, Ur­banowski, and Green­berg iden­ti­fied genes in­volved in this kind of self­/not-self recog­ni­tion. They as­sayed 3600 Pro­teus mu­tants made by trans­po­son mu­ta­ge­n­e­sis for their abil­ity to make bound­aries with ad­ja­cent neigh­bors. They found a mu­tant that not only forms bound­aries with their par­ents (thus dis­own­ing its an­ces­try), but with all other mu­tants as well. This mu­tant has lost the abil­ity to rec­og­nize self. At the bound­ary of two in­com­pat­i­ble strains, one finds a few cells from both strains, some of which are rounded up, sug­gest­ing per­haps cell dam­age by a mech­a­nism as yet un­known. The same group of re­searchers re­cently mapped the mu­ta­tions to a six gene operon. What turns it on? Could it be ac­ti­vated by con­tact with the agar sur­face? Not known yet.

The au­thors called the genes in­volved ids, for IDen­tifi­ca­tion of Self. Three of these genes (idsB, idsC, and idsF) en­code func­tions es­sen­tial for self-recog­ni­tion while two (idsE and idsF) en­code iden­tity de­ter­mi­nants. The pro­tein prod­ucts of only two of these genes, IdsA and IdsB, have sig­nif­i­cant se­quence sim­i­lar­ity to other bac­te­r­ial pro­teins, these both be­ing part of a gene clus­ter in­volved in type VI se­cre­tion in Vib­rio cholerae. Not much help there as yet. Other uniden­ti­fied de­ter­mi­nants may also play a role here. Look­ing at five sep­a­rate iso­lates of P. mirabilis some of the genes in the ids operon turn out to be highly con­served, oth­ers, such as idsD and idsE, quite vari­able.

The story is ac­tu­ally quite in­volved. The au­thors made a GFP fu­sion with the IdsA pro­tein and found that the ids pro­moter shows a bi­modal ex­pres­sion. In other words, some cells at the ad­vanc­ing edge of the colony lit up un­der the flu­o­res­cence mi­cro­scope, oth­ers did not. This sug­gests that there is a bistable switch that con­trols ids tran­scrip­tion. Only those cells ex­press­ing the ids genes are able to sense self and non-self. What this means must await an­other day. In­trigu­ing is that cells grown in broth (swim­mers, not swarm­ers) ex­hib­ited this bi­modal ex­pres­sion, as well; a solid sur­face is not needed. Keep in mind that the tran­scrip­tion was from an idsA-gfp con­struct on a plas­mid, so, as the au­thors point out, it re­mains to be seen if this hap­pens with a chro­mo­so­ma­lly-lo­cated pro­moter. The topic of bista­bil­ity is get­ting a fair amount of trac­tion in the bac­te­r­ial world. If you can't wait and want to get a whiff of the cur­rent think­ing, see this com­men­tary by David Dub­nau.

Bi­modal ex­pres­sion of the idsA-gfp fu­sion in cells at the outer edge of a swarm. (Left) phase im­age; (Cen­ter) flu­o­res­cence im­age; (Right) false-col­ored im­age with the agar back­ground in red, flu­o­res­cence bright cells in green, and dark cells in black. As­ter­isks = ex­am­ples of dark cells. Ar­row­heads = swarm tracks. Bar = 50 μm. Source

An­other find­ing of in­ter­est was that ex­pres­sion of the ids operon in­creases in late log­a­rith­mic and early sta­tion­ary phases. Could this be reg­u­lated by quo­rum sens­ing, one asks? Also, ids ex­pres­sion in­creases when a swarm of non-self cells ap­proaches. To test whether this might be due to some non-self cells wan­der­ing across the bound­ary, the re­searchers placed a bac­te­ria-re­tain­ing fil­ter be­tween the two swarms. This blocked the ef­fect, so cell-to-cell con­tact, or the pres­ence of a non-dif­fusible sub­stance, seems to be in­volved. For sev­eral rea­sons, a toxin-an­ti­toxin mech­a­nism does not seem to play a role here, mainly be­cause recog­ni­tion does not lead to cell death, nor do known bac­te­ri­ocins of P. mirabilis.

Tan­ta­liz­ing ques­tions re­main. What are the prod­ucts of the ids genes? How do they work? Rec­og­niz­ing non-self is one thing; act­ing on that iden­ti­fi­ca­tion is an­other. How is all this done? This phe­nom­e­non is not lim­ited to Pro­teus species. Other bac­te­ria, e.g., Pseudomonas, swarm with gusto and may well use such self­/non-self recog­ni­tion mech­a­nisms. We en­vis­age that a lot of work of this sort will come forth and that we may be­come fur­ther en­light­ened about the so­cial go­ings-on hap­pen­ing on the sur­face of an agar plate. And, no doubt, we'll be­come fur­ther en­light­ened about the many other ways that mi­crobes in­ter­act.

Post-Pub­li­ca­tion Note: Since I de­cided to pay at­ten­tion to Pro­teus mirabilis, it de­cided to rec­i­p­ro­cate. I had a mild uri­nary tract in­fec­tion re­cently, and guess what grew out: P. mirabilis! We're even.

Ref­er­ence

Gibbs KA, Wen­ren LM, & Green­berg EP (2011). Iden­tity gene ex­pres­sion in Pro­teus mirabilis. Jour­nal of Bac­te­ri­ol­ogy, 193 (13), 3286−3292. PMID 21551301