Borrelia and it's not-so-loose ends (2|2)

by Christoph
 

I wrote in the first part that "in the the se­cond part I will con­si­der their ex­tra­va­gant pe­ri­plas­mic(!) fla­gel­la. There will be no "loose ends" left in ei­ther part, I pro­mise." Spoi­ler: no "loose ends" but a (know­ledge) gap in the middle, that is, when it comes to cell di­vis­ion in Bor­re­lia. Here it goes...
 

Figure 2.1  A Representative tomo­graph­ic slice of the tip of a wild-type (WT) Bor­re­lia cell. B Sur­face view of the cell tip. The mo­tors are co­lor­ed in red. Scale bar 100 nm. Pe­ri­plas­mic fla­gel­la (PF) are co­lor­ed in pur­ple. C Sur­face views of a half of the cell. Scale bar 250 nm. The PF from the oth­er tip are co­lor­ed in yel­low. D Sur­face views of the full WT cell. Scale bar 500 nm. Source

Endoflagella, also called 'axi­al f­ila­ments' or 'pe­ri­plas­mic fla­gel­la', "are an­chor­ed at each end (pole) of the bac­te­ri­um with­in the pe­ri­plas­mic space (bet­ween the in­ner and out­er mem­bran­es) where they proj­ect back­wards to ex­tend the length of the cell. These cause a twist­ing mo­tion which al­lows the spi­ro­chae­te to move about" says Wi­ki­pe­dia in the en­try for Spi­ro­chae­tes. The over­all ar­chi­tec­ture of en­do­fla­gel­la − in­ner‑mem­brane em­bed­ded mo­tor rings, hooks, fla­gel­lar fi­la­ments − is not no­tab­ly dif­fer­ent from that of other fla­gel­la. Ex­cept that they don't pos­sess the out­er-mem­brane rings and do not form bund­les, a com­mon phe­no­men­on for mul­tiple po­lar fla­gel­la, but are ar­ranged side-by-side as a belt (F­igure 2.1).
 

This belt-like arrangement of the Bor­rel­ia en­do­fla­gel­la and their ro­tat­ion as a belt re­sults in a con­si­der­able tor­que on the 'pro­to­plas­mic cell cy­lin­der' (the most fre­quent­ly used term in re­search pa­pers but I would pre­fer to call it a 'pro­to­plas­mic tube' ra­ther, em­pha­siz­ing its mi­nute cross sect­ion.) This tor­que and the re­sist­an­ce of the 'pro­to­plas­mic tube' to­gether de­ter­mine the cell shape and makes the cells move in a corks­crew-like fash­ion. In­deed, fla­gel­la-less mu­tants ap­pear flat­tened-out and don't move.
 

In case you're unhappy with the sta­tic 2D pic­tur­es in Fi­gure 2.1 you may want to take a 3D-ride through a Bor­rel­ia burg­dor­fe­ri cell here in 'The At­las of Bac­te­ri­al & Ar­chae­al Cell Struc­ture'. I'm temp­ted to warn you "fas­ten your belt!", the Bor­re­lia sure did.
 

What happens when flagella ema­na­ting from both cell ends have wrap­ped as a belt around the 'pro­to­plas­mic tube' all their way to mid­cell? It has been found that the fla­gel­la over­lap with each other as to give a belt that runs through the en­tire cell length (see Fi­gure 21.1D). This makes the cork­screw-like mo­ve­ment of Bor­re­lia cells through me­dia with low­er and like­wise with high­er vis­co­si­ty (at low Rey­nolds num­bers!) very ener­gy sav­ing, thus ef­fi­cient, and no less ef­fi­cient when crawl­ing on mi­ni­mal­ly wet­ted sur­fac­es like host tis­sue or agar plates. (A prac­ti­cal hint: If you ever get into quick­sand − better you don't! − just don't start fran­ti­cal­ly row­ing with your arms but re­call how Bor­re­lia does it: move cork­screw-like.) A tricky quest­ion for which there is no con­clus­ive ans­wer yet: how do the two fla­gel­lar mo­tor pat­ches "com­mu­nicate" with each other to make sure that one set of pat­ches sets the fla­gel­la in­to clock­wise ro­ta­tion while the other in­to coun­ter-clock­wise ro­ta­tion? This is ne­ces­sa­ry to achie­ve a con­sist­ent di­rect­ion of swim­ming for a cell − if both sets of fla­gel­la ro­ta­te in the same di­rect­ion the cell stops mov­ing and gets stuck with a "kink" in its middle (which is oc­ca­sion­al­ly ob­ser­ved.) Or can the mo­tor pat­ches do away with "com­mu­ni­cat­ing"­ and re­ly in­stead on a subtle gra­dient of sig­nal­ing mo­le­cules over their con­si­der­able length? We don't know.
 

Figure 2.2 Cryo-ET tomo­gram of a B. burg­­dor­fe­ri cell. Che­mo­re­cep­tor ar­ray (blue), fla­gel­lar mo­tor (yel­low), Out­er mem­­brane ve­sicle-chain (pink). Scale Bar 100 nm. (Click ima­ge for the un-an­no­tat­ed ori­gi­nal.) Cour­te­sy of Ari­a­ne Brie­gel

What determines the di­rect­ion of ro­ta­tion of the fla­gel­la or its re­vers­al? Just like in other fla­gel­lat­ed bac­te­ria: sig­nal tran­sduct­ion via che­mo­sen­so­ry ar­rays. These ar­rays "per­ceive en­vi­ron­men­tal sti­mu­li and col­lec­ti­ve­ly use them to con­trol the phos­pho­ry­la­tion le­vel of re­cei­ver pro­teins (CheY and CheB), which me­di­ate mo­tor con­trol and sen­so­ry adap­ta­tion, re­spec­ti­ve­ly." To break this down a bit: a sig­nal (a su­gar or an ami­no acid) that is tem­po­ra­ri­ly bound by the pe­ri­plas­mic do­main of its cog­nate (in­ner-)mem­brane-bound re­cep­tor (MCP) is "pick­ed up" as a phos­pha­te group by CheW, which pas­ses it on to CheA. CheA in turn then phos­pho­ry­lat­es CheY and CheB. CheY~P binds to the mem­brane-em­bed­ded fla­gel­lar mo­tor and slows down its ro­ta­tion, lead­ing to move­ment re­vers­al if enough "bra­kes" are ap­plied. CheY ra­pid­ly loos­es its phos­phate group by de-phos­pho­ry­la­tion, thus re­stor­ing its ini­tial state. CheB~P leads to the re­sto­ra­tion of the re­cep­tor's "rea­dy" state via an in­ter­play with CheR and a me­thyl-group ex­change on the MCP. The ef­fi­cien­cy of this "phos­pho­re­lay" is ki­ne­ti­cal­ly con­trol­led for on/off-swit­ch­ing, and the over­all sig­nal strength mar­ked­ly am­pli­fied by the close pro­xi­mi­ty in the ar­ray of the pro­teins in­volv­ed.
 

You see in Figure 2.2 a che­mo­sen­so­ry ar­ray of Bor­re­lia in side view, pat­ched to and part­ly stick­ing with­in the in­ner mem­brane (via the MCPs), al­most like a band-aid. Its lo­ca­tion is in close pro­xi­mi­ty to the line-up of fla­gel­lar mo­tors at the gra­du­al­ly nar­row­ing cell end. There is vir­tu­al­ly no way to make the trans­fer of the pe­ri­plas­mic sig­nal across the cy­to­plasm via CheY to the fla­gel­lar mo­tor any shor­ter! In the top view, the band-aid ap­pear­ance of a che­mo­sen­so­ry ar­ray is even more clear­ly vi­sible, here for Tre­po­ne­ma as I had no sample from Bor­rel­ia avai­lable but both spi­ro­chae­tes cells have this nar­row cross-section of <0.5 µm. The he­xa­gon­al grid of the ar­ray, the 'base­plate', is form­ed by the CheA and CheW pro­teins, and is struc­tu­ral­ly high­ly adap­tab­le to vary­ing mem­brane cur­va­tures. On top of this, the he­xa­gon­al grid-struc­ture of the ar­ray has ano­ther ex­qui­site pro­per­ty: it can be ex­pand­ed or shrunk on all sides at any time by self-as­sem­bly/dis-as­sem­bly, not un­like phage cap­sids. Keep in mind that just as the Bor­rel­ia have fla­gel­la at both cell ends, they also have the ar­rays at both.
 

These two 'cell ends' of the Borrelia as you see them in Fi­gur­es 2.1 and 2.2 look more like point­ed co­ni­cal hats and not even re­mo­te­ly like the half-dome shap­ed 'cell poles' that you're fa­mil­iar with from nor­mal rod‑shaped bac­te­ria like B. sub­ti­lis or E. co­li, or coc­coid bac­te­ria like Strep­to­coc­cus pneu­mo­niae. Spi­ro­chae­tes di­vide by bi­na­ry fis­sion − ea­si­ly ob­served in the mi­cro­sco­pe − but the mo­le­cu­lar or­ches­tra­tion of sep­tum for­ma­tion and cell di­vi­sion of Bor­re­lia and other spi­ro­chae­tes is poor­ly un­der­stood. Yes, Bor­rel­ia en­codes the FtsZ and MreB cy­to­ske­le­tal pro­teins but their pre­cise roles are not known.
 

Figure 2.3  Population ana­ly­sis of HA­DA sig­nal du­ring the B. burg­dor­fe­ri cell B31 MI cycle. A (left) De­mo­graph ana­ly­sis of HA­DA fluor­es­cen­ce pro­fil­es in which all B31 MI cells in the po­pu­la­tion (n=276) were or­gan­iz­ed in as­cend­ing cell length or­der (left to right). The heat map dis­plays the re­la­ti­ve fluor­es­cen­ce in ar­bi­tra­ry units (0–1). A (right) Re­pre­sen­ta­ti­ve fluor­es­cen­ce ima­ges of cells (I, II, and III) at dif­fer­ent points of the cell cycle (dash­ed ar­rows) are de­pict­ed on the right with cell boun­da­r­ies in yel­low. B Scat­ter plot show­ing the po­si­tions of HA­DA zone po­si­tions as a funct­ion of cell length. C Same as in B, ex­cept that zone po­si­tions were plot­ted in cel­lu­lar co­or­di­nat­es. (Click ima­ge for the com­ple­te fi­gu­re.) Source

A recent study from Chris­ti­ne Ja­cobs-Wag­ner's lab came clos­er to an un­der­stand­ing of cell growth and di­vi­sion in Bor­rel­ia by using pep­ti­do­gly­can syn­the­sis as pro­xy. Pep­ti­do­gly­can (PG), the thin 'sup­port stock­ing' po­ly­mer that en­ti­re­ly co­vers the mem­brane-bound­ed cy­to­plasm, is con­stant­ly syn­the­siz­ed du­ring cell growth by cross­link­ing su­gar oligo­mers with short pep­tid­es in the pe­ri­plasm. The short cross­link­ing pep­tid­es con­tain the un­usu­al ami­no acid D-ala­nine and the au­thors used a fluor­es­cent D-ala­nine de­ri­va­tive, 7‑hy­dro­xy­cou­ma­rin‑ami­no‑D‑ala­nine (HADA), to mo­ni­tor PG syn­the­sis in Bor­re­lia by fluor­es­cen­ce mi­cro­sco­py. In grow­ing cul­tur­es con­tain­ing cells of all lengths, they found HADA in­cor­po­ra­tion through­out the length of the cells with slight­ly de­creas­ing sig­nal in­ten­si­ties at the cell ends. Stun­ning, how­ever, was the con­cen­tra­tion of strong HADA sig­nals in the form of zones (of up to 3 µm!) ra­ther than de­fined fo­ci at mid­cell and, in ad­di­tion, at the 1/4 and 3/4 po­si­tions re­la­tive to the cells' lengths (F­igure 2.3A). Note that all 276 cells in the 'de­mo­graph' shown in Fi­gure 2.3 were "straight­end" by soft­ware prior to alig­ning them by size (Figure 2.3A) and for the other eva­lua­tions (Fig.2.3B,C).
 

It is a tho­rough­ly con­firm­ed ob­ser­va­tion yet with­out an un­der­stand­ing of the un­der­ly­ing (mo­le­cu­lar) me­chan­ism that the sig­nal zones at the 1/4 and 3/4 po­si­tions ap­pear at the time of ma­xi­mum sig­nal in­ten­si­ty in the cen­ter of the cell (stage II). How­ever, it in­di­cates that fu­ture cell di­vis­ion sites are al­rea­dy "tapped" if the in­ten­sit­y at these po­si­tions in­creas­es at a time when the sig­nal in­ten­si­ty of the mid­cell zone de­creas­es to ze­ro, in­di­cat­ing cell di­vis­ion at this point there (stage III). Who tells the "di­vis­ion ma­chi­ne­ry" that the rep­li­cat­ed chro­mo­so­me plus the rep­li­cat­ed plas­mid zoo has been safe­ly se­gre­gat­ed to their re­spec­tive cell hal­ves? As I said above, no "loose ends" for Bor­re­lia but still some nag­ging quest­ions at midcell.