Space-tol­er­ant Mi­cro­nauts

by Alise Muok and Ar­i­ane Briegel

Try to imag­ine a crea­ture that can sur­vive from the heat of 150°C (300°F) to the cold of –270°C (–454 °F), in the ab­sence of oxy­gen, af­ter com­plete de­hy­dra­tion, at 1,000× at­mos­pheric pres­sure, and af­ter be­ing ex­posed to the ra­di­a­tion and vac­uum of space. Who might such space trav­el­ers be? Given their wide­spread news cov­er­age, in­clud­ing a re­cent post by Elio, your first thought might be tardi­grades. But there is a dif­fer­ent type of crea­ture that can also sur­vive the ex­treme con­di­tions of space.

Fig­ure 1. Tar­dinaut and spore com­pan­ion, by Alise Muok

That's right, we are talk­ing about bac­te­r­ial spores! Like tardi­grades, many bac­te­ria re­spond to stress con­di­tions by go­ing into a dor­mant yet re­silient state that can re­vert back into an ac­tive state when their sur­round­ings be­come more fa­vor­able. While tardi­grades un­dergo the­se tran­si­tions by reg­u­lat­ing spe­cific metabo­lic processes, bac­te­ria pro­duce spores that can germi­nate. These spores con­tain the iden­tical ge­netic in­for­ma­tion of their par­ent cell but they are coat­ed in a thick layer of pep­ti­do­gly­can (PG) and pro­teins that pro­tect them from the envi­ron­ment. When envi­ron­men­tal con­di­tions are met, the spore PG coat will dis­as­sem­ble to pro­duce an ac­tive bac­te­r­ial cell once again. Be­cause of their pro­tec­tive coat, the spores can sur­vive in en­vi­ron­ments the par­ent cell could not, in­clud­ing the harsh con­di­tions of space.

The re­arrange­ment of PG lay­ers dur­ing sporu­la­tion is a com­plex process that has been stud­ied ex­ten­sively in the bac­te­r­ial model or­gan­ism Bacil­lus sub­tilis by mi­croscopy tech­niques, which al­low re­searchers to di­rectly view and im­age PG lay­ers in vivo. Us­ing these data, re­searchers have seen that sporu­la­tion oc­curs in four sta­ges. In the first and sec­ond stages, the mother cell repli­cates its genome and then asym­met­ric septa­tion of the cell oc­curs to sep­a­rate the genomes into two diffe­rent com­part­ments – the smaller of these will even­tu­ally be­come the spore. In the third stage the sep­tum starts to curve around the form­ing spore (called the fore­s­pore) un­til it is com­pletely en­gulfed in­side the mother cell (see here in STC). In the fi­nal stage, the fore­s­pore be­comes surroun­ded by thick lay­ers of PG and then is re­leased from the mother cell. How­ever, sev­eral as­pects of this sys­tem have re­mained poorly un­der­stood due to lim­i­ta­tions of mi­croscopy tech­niques. In an ex­cit­ing leap for­ward to un­der­stand­ing this process, new mi­croscopy data pre­sented in Khanna et al. (Open Ac­cess PDF) elu­ci­date im­por­tant fea­tures of PG syn­the­sis and de­gra­dation dur­ing sporu­lation in B. sub­tilis. This ad­vance was made pos­si­ble by the use of a tech­nique called cryo-elec­tron mi­croscopy (cryo-EM) that al­lowed these re­searchers to im­age sporu­lat­ing cells at the high­est re­solution re­ported thus far.

Fig­ure 2. Slices through cryo-elec­tron tom­o­grams rep­re­sent­ing dif­fer­ent stages of engulf­ment: D flat po­lar sep­tum (Stage IIi), F curved sep­tum (Stage IIii). Scale bar 200 nm. The cor­responding fore­s­pore mem­brane (green) and the mother cell mem­brane (yel­low) are anno­tated in E,G res­pectively. n num­ber of tomo­grams ac­quired for each cell type. Scale bars have been omit­ted for E,G as cells are shown in pers­pective views. Source

Be­fore the in­ven­tion of elec­tron mi­cro­scopes, sci­en­tists were able to see liv­ing things with light mi­cro­scopes, but could not see in­side cells at a mol­e­c­u­lar res­o­lu­tion due to the lim­it­ing wave­length of vis­i­ble light. How­ever, the wa­ve­length of an elec­tron is about 100,000× shorter than that of vis­i­ble light, re­veal­ing struc­tural de­tails at an un­pre­cedented level. To vi­su­al­ize the power of an elec­tron mi­cro­scope in your mind's eye, imag­ine that we got so frus­trated with wri­ting this ar­ti­cle that we asked our friends at NASA to shoot it to the sur­face of the moon (next to the dor­mant wa­ter bear colony lo­cated in the 'sea of seren­ity', 32.595°N, 19.349°E). An elec­tron mi­croscope has enough mag­ni­fy­ing power that we would still be able to read the ar­ti­cle from here.

While elec­trons have a short wave­length that makes them ideal for high-res­o­lu­tion imag­ing, the elec­trons can only be used for imag­ing if the sam­ple is con­tained in a vac­uum, thus pre­vent­ing the use of any sam­ple that con­tains wa­ter – which ba­si­cally means any­thing bi­o­log­i­cal. In the past, this is­sue was cir­cum­vented by the de­vel­op­ment of elab­o­rate sam­ple prepa­ra­tion work­flows that typ­i­cally in­cluded sam­ple de­hydration, heavy metal stain­ing, plas­tic em­bed­ding, and sec­tion­ing. This al­lowed imag­ing of a wide va­ri­ety of bi­o­log­i­cal speci­mens and al­lowed re­searchers to gain great in­sight into cel­lu­lar bi­ol­ogy. How­ever, the in­vasive na­ture of the prepa­ra­tion pro­ce­dures af­fects the del­i­cate ultra­struc­ture of cells and pre­vents more de­tailed in­sight into struc­tures at the macro­mol­e­c­u­lar level. But then cryo-EM came to the res­cue! In­stead of in­vasive prepa­ra­tion pro­ce­dures, bi­o­log­i­cal sam­ples are sim­ply plunged into a liq­uid cryo­gen. Freez­ing of the sam­ple hap­pens so fast that wa­ter mol­e­cules are un­able to form crys­tals, and the spec­i­mens are em­bed­ded in a glass like, vit­re­ous ice. Un­like crys­talline ice, vit­re­ous ice does not dam­age the sam­ple in any way so the bi­o­log­i­cal sam­ples will re­tain na­tive macro­mol­e­c­u­lar struc­tures. Fur­ther­more, to study mi­crobes in three di­men­sions by cryo-EM, we can sim­ply tilt our spec­i­mens in the mi­cro­scope while ta­king mul­ti­ple im­ages with dif­ferent ori­en­ta­tions of the sam­ple rel­a­tive to the elec­tron beam and then com­pu­ta­tion­ally back-project these im­ages to gen­er­ate a 3D im­age. This is sim­i­lar to do­ing a CAT scan, but in this case we ro­tate the sam­ple and not the mi­cro­scope. This method is called cryo-elec­tron to­mography (cryo-ET) and has emerged as a pow­er­ful tool to study the struc­ture and func­tion of macro­mo­le­cu­lar com­plexes in­side of cells.

Fig­ure 3. Slice through a cryo-elec­tron to­mo­gram of wild type B. sub­tilis spo­rangium. E,F An­no­tated fore­spore (green) and mother cell (yel­low) mem­branes for the tomo­gram shown in D as viewed from both the left (blue *) and the right (ma­roon *) sides respec­ti­vely, with in­sets of zoomed-in views of the lea­d­ing edge of the en­gulf­ing mem­brane of both si­des. This la­bel­ing scheme is fol­lowed through H–N. G Schematic show­ing the lo­cal­iza­tion of DMP PG deg­radation ma­chin­ery (yel­low pac­man) and PG syn­thases (blue cir­cles). Membra­nes (red), lat­eral PG (gray), sep­tal PG (pink) and new PG (pur­ple) are also high­lighted. H Slice through a cryo-elec­tron to­mo­gram of spoIIP mu­­tant spo­rangium. I,J An­notated mem­branes for the to­mo­gram shown in H with in­sets of zoomed-in views of the lead­ing edge of the en­gulfing mem­brane of both sides. K Schematic rep­re­sent­ing a cell in which the DMP com­plex (yel­low pac­man) does not as­sem­ble. L Slice through a cryo-elec­tron to­mo­gram of cephale­xin-treated spo­rangium. M,N An­notated mem­branes for the to­mo­gram shown in L with in­sets of zoomed-in views of the lead­ing edge of the en­gulfing mem­brane of both sides. O Sche­matic rep­re­sent­ing a cell in which PG syn­the­sis (blue cir­cles) has been in­hib­ited. Scale bar for D,H,L 200 nm. Scale bars not shown for sur­face ren­dered im­ages ow­ing to their per­spec­tive na­ture. Source

Be­fore the Khanna et al. pa­per, re­search pub­lished by To­cheva et al. in 2013 also uti­lized cryo-ET to pro­vide great in­sight into the process of sporu­la­tion in B. sub­tilis. They not only saw all the stages of sporu­la­tion in this or­gan­ism but also de­duced that a thin PG layer re­mains around the fore­spore dur­ing en­gulf­ment. It had been pre­vi­ously thought that the PG layer would com­pletely dis­as­sem­ble to accommoda­te the bend­ing force needed for engulf­ment. How­ever, the res­o­lu­tion achieved in that study was not suf­fi­cient to un­ra­vel finer struc­tural de­tails. This li­mi­ta­tion re­sulted from the fact that spores and whole bac­te­rial cells are very large ob­jects for cryo-ET, which is ra­ther lim­ited by the thick­ness of sam­ples that can be ima­ged. Just like with light mi­croscopy, if your sam­ple is very thick, ob­jects will just ap­pear as dark sil­hou­ettes with­out any in­ter­nal de­tail. In­stead, the elec­trons must be able to pen­e­trate through the sam­ple to pro­vide an im­age. In prac­tice, sam­ples of around half of a mi­crom­e­ter or less are suit­able, while a thicker spec­i­men is chal­leng­ing or even im­pos­si­ble.

Spores are one to two mi­crom­e­ters in di­am­e­ter, and sur­rounded in a dense PG coat… So how was the team of Khanna et al. able to un­cover the finer de­tails of spore for­mation in B. sub­tilis us­ing cryo-ET? They were able to achieve this by us­ing an­other new in­stru­ment in the cryo-EM tool­box called 'cryo-fo­cused ion beam milling' (cryo-FIB). Es­sentially, this method lets you use an ion beam that is so pre­cise that you can cut cells into lay­ers that are thin enough for cryo-ET ex­per­i­ments. Af­ter vit­ri­fy­ing thicker sam­ples such as whole B. sub­tilis cells un­der­go­ing spo­rulation, the sam­ple is loaded in the cryo-FIB instru­ment. Here, a gal­lium ion beam is used to cut thin 'win­dows' into the oth­er­wise too thick sam­ple. The sam­ple is then trans­ferred into a cryo-EM scope, where to­mo­grams (us­ing cryo-ET) of high qual­ity can be col­lected at the po­si­tions of these 'win­dows'. So to put things more pre­cisely, Khanna et al. didn't just use an elec­tron mi­cro­scope to elu­ci­date sporu­la­tion at un­par­al­leled de­tail, they first used cryo-FIB to make their sam­ples thin­ner and then used cryo-ET to gen­er­ate 3D mod­els of these thin slices (Fig­ures 2+3).

Us­ing this work­flow, Khanna et al. re­vealed the or­ga­ni­za­tion of PG around the fore­s­pore in greater de­tail. Specif­i­cally, they were able to see that the PG layer is con­tin­u­ously syn­the­sized and degrad­ed at the lead­ing edge of the mother cell dur­ing en­gulf­ment. This al­lows the sep­tum to be 'push­ed' around the fore­s­pore and sup­ply the force needed for en­gulf­ment. Fur­ther­more, the resolu­tion of these data was so high that they were able to re­solve tiny fin­ger-like pro­jec­tions of the moth­er cell mem­brane at the lead­ing edge that forms around the fore­s­pore. Their for­ma­tion de­pends on an en­zyme com­plex called DMP. It is formed by the pro­teins SpoIID, SpoIIM and SpoIIP, which to­gether have PG de­grad­ing ac­tiv­ity. This com­plex is teth­er­ing the mem­brane to the PG and de­grad­ing it ahead of the en­gulf­ing mem­brane. The au­thors fur­ther sug­gest a func­tional role of the fin­ger­like pro­tru­sions, which may pro­vide the mem­brane a tighter lat­eral grip dur­ing its move­ment by serv­ing as a ratchet that pre­vents back­ward slid­ing.

Since the rel­a­tively re­cent in­tro­duc­tion of cryo-EM, emerg­ing meth­ods have in­creas­ingly lead to as­tounding dis­cov­er­ies of com­plex bi­o­log­i­cal processes. We can now see in­side or­gan­isms that are lite­rally frozen in time in de­tails we pre­vi­ously thought im­pos­si­ble. And for all this we need to thank the high-tech process that is cryo-EM imag­ing. Imag­ine, your sam­ples are not just put un­der a lens to mag­nify your vi­sion, but they are put in­side a vac­uum sealed col­umn, at tem­per­a­tures be­low –170°C, and then ex­posed to high amount of ra­di­a­tion from an elec­tron source to gen­er­ate an im­age that ex­poses the func­tion of mol­e­cules. Thank you, cryo-EM! And since these con­di­tions are very sim­i­lar to those in space, it makes us won­der if wa­ter bears and bac­te­r­ial spores could be 're­vived' af­ter imag­ing them in a cryo-EM scope. So the next time you see fan art of a wa­ter bear in space, imag­ine a bac­te­r­ial spore friend in ac­com­pa­ni­ment!

 

Ar­i­ane is a pro­fes­sor at Lei­den Uni­ver­sity in the Nether­lands. She has nearly 20 years of ex­pe­ri­ence us­ing cryo-elec­tron mi­croscopy to study the ul­tra­struc­ture of mi­crobes. Her lab fo­cuses on investi­gat­ing how mi­crobes sense and re­spond to their en­vi­ron­ment and ex­plores the struc­tures and func­tions of the mol­e­c­u­lar com­plexes in­volved in these be­hav­iors. Alise is a post­doc in Ar­i­ane Briegel's lab at Lei­den Uni­ver­sity. She has a back­ground in struc­tural bio­logy, specif­i­cally in analysing pro­tein sys­tems in bac­te­ria. When they are not in lab, Alise loves to paint and prac­tice aer­ial silks. Ar­i­ane loves gar­den­ing, hik­ing or learn­ing how to do aer­ial silks from Alise!