Fairy Lights (nanome­ter scale)

Pic­tures Con­sid­ered #46

by Christoph

This post fea­tures elec­trons, which are among the small­est things pos­si­ble. There are, of course, even much smaller things than elec­trons but this blog keeps clear of the quan­tum realm, most of the time. Yet to make up for the elec­trons' teen­sy­ness – if they could keep still some 4 × 10²¹ would fit into the pe­riod at the end of this sen­tence – they are fea­tured here in two roles. When acce­le­ra­ted at 200 kV in a trans­mis­sion elec­tron mi­cro­scope (TEM) the wave­length of elec­trons comes close to 2.5 pi­come­ters and al­lows for the vi­su­al­iza­tion of macro­mol­e­c­u­lar and sub­cel­lu­lar struc­tures in the sub‑nanometer range. Such struc­tures can­not be re­solved by con­ven­tional light mi­croscopy that is con­strained to re­solv­ing struc­tures in the ~200 nm range (see here in STC for more). So, by elec­tron mi­croscopy, elec­trons can help elu­ci­date the struc­ture of outer mem­brane ex­ten­sions (OME) that some bac­te­r­ial cells use to shut­tle… elec­trons, you guessed it. Spoiler: there's no way to see elec­trons in per­sona, but at least some in fairy lights, al­most (Fig­ure 1).

Fig­ure 1. 3D iso­sur­face view of the outer mem­bra­ne ex­tension (OME) in Fig. 2 A with all of the in­te­rior and ex­te­rior den­si­ties (pu­ta­tive MtrA + MtrC, respec­tively) re­presented as model points in green and red, respecti­vely (Scale bar: 50 nm). Screen­shot from Movie S12, at min 1:32. Source

When bac­te­r­ial cells need en­ergy they usu­ally em­ploy two bio­chem­i­cal path­ways, gly­col­y­sis and res­pi­ra­tion that read, in short and ne­glect­ing ex­act sto­i­chiom­e­try: C₆H₆O₆ + O₂ → CO₂ + H₂O. Well hid­den in this chem­i­cal equa­tion is what ac­tu­ally dri­ves en­ergy con­ver­sion: 4 H⁺ + 4 e^– + O₂ → 2 H₂O. And the key term here is: 4 H⁺ + 4 e^–, that is, the en­zy­matic sep­a­ra­tion of hy­dro­gen ions (H⁺) from elec­trons (e^–). The hy­dro­gen ions are pumped out of the cyto­plasm to build up an elec­tro­chem­i­cal gra­di­ent across the in­ner mem­brane that pow­ers the cell's dy­namo, ATP syn­thase. While re­plen­ish­ing their ATP pool the cells have to dis­pose of the elec­trons, which do not sim­ply "dif­fuse away". Again with­out giv­ing the ex­act sto­i­chiome­tries, these elec­trons are fun­neled through sev­eral in­ner-mem­brane com­plexes to­wards com­plex IV (cy­tochrome c oxi­dase). This en­zyme cat­alyzes their re­ac­tion with O₂ to yield first [O₂]· rad­i­cals, and as end­prod­uct H₂O. Thus oxy­gen (O₂) serves as elec­tron ac­cep­tor and al­lows bac­te­ria breathe with ease. Anaer­obe bac­te­ria can "breathe" by us­ing other sol­u­ble elec­tron ac­cep­tors, as for ex­am­ple ni­trate (NO₃) or sul­fate (SO₄). This, of course, re­quires dif­fer­ent en­zymes for the last step. Trick­ier is the use of in­sol­u­ble elec­tron ac­cep­tors, for ex­am­ples metal ox­ides such as man­ganese ox­ide or fer­ric iron (Fe³⁺)-con­tain­ing com­pounds. A num­ber of bac­te­ria from dif­fer­ent phyla have turned mas­ter elec­tri­cians and vir­tu­ally "plug ca­bles into a socket" of such met­als, that is, cel­lu­lar ex­ten­sions termed 'mi­cro­bial nanowires', which were in­tro­duced to STC back in 2008.

Fig­ure 2. Po­si­tions and pack­ing of dec­a­heme cytochro­mes (deca=10) along the outer mem­brane (OM) exten­sion length in S. onei­den­sis. A elec­tron cry­oto­mog­ra­phy (ECT) im­age of an un­fixed OM extens­ion show­ing den­si­ties on both the in­te­rior and ex­te­rior of the OM corres­pon­­­ding to pu­ta­tive MtrA and MtrC cyto­chromes, re­spec­tively (Scale bar: 50 nm). B1 en­larged view of boxed area from A. B2 com­pa­ri­son of EM den­si­ties in B1 with the crys­tal struc­ture of MtrC, low-res­o­lu­tion SAXS model of MtrA, and the MtrB ho­molog LptD, high­light­ing the si­milarity in over­all shape and size of these struc­tures to the ob­served EM den­si­ties. Red, MtrC crys­tal struc­ture; green, sur­face view of MtrA SA­XS model; blue, LptD crys­tal struc­ture; dot­ted green, out­line of pu­ta­tive MtrA den­si­ties on the EM map (Scale bar: 10 nm). C a 3D iso­sur­face view of the OM ex­ten­sion in A with all of the in­terior and ex­te­rior den­si­ties (pu­ta­tive MtrA and MtrC, re­spec­tively) rep­re­sented as model points in green and red, re­spec­tively (Scale bar: 50 nm) (see Movie S12) D–E rep­re­sen­ta­tive re­gions from the OM ex­ten­sion in A demon­strat­ing dif­fe­rences in pack­ing den­sity of MtrA and MtrC. Con­tin­u­ous ex­te­rior (D1) and in­te­rior (E1) den­si­ties that may be re­lated to tightly packed MtrC and MtrA, re­spec­tively. D2 and E2 are dupli­ca­tes of D1, E1, re­spec­tively, with model points or lines high­light­ing the in­te­rior (green) and ex­te­ri­or (red) den­si­ties (Scale bars: 20 nm). Source

Among the first bac­te­ria that were stud­ied for their abi­li­ty to "breathe metal" and "grow on elec­trodes" was She­wa­nella onei­den­sis MR‑1. An ear­lier study had al­ready con­clu­sively shown, by com­bin­ing con­duc­tiv­ity measure­ments and atomic force mi­croscopy (AFM), that this ca­pa­city de­pends on cel­lu­lar ap­pendages, the no­to­ri­ous 'nano­wires'. Now a re­cent study by the El-Nag­gar and Jensen labs re­vealed more struc­tural de­tails of these 'appen­da­ges', and iden­ti­fied them as outer mem­brane ex­ten­sions (OMEs) (Fig­ure 1). Their method in­cluded the fol­low­ing steps, briefly: 1. grow S. onei­den­sis on a metal grid un­der anaer­o­bic con­di­tions to pro­mote the pro­duc­tion of OMEs and in the pres­ence of a (non-toxic) flu­o­res­cent membra­ne stain, 2. fix the cells and their OMEs at var­i­ous time points by treat­ment with glu­taralde­hyde, 3. plunge-freez­ing of the grids to pre­vent the for­ma­tion of ice crys­tals that would dis­tort or de­stroy sub­cel­lu­lar struc­tures, 4. mo­nitor the sta­tus of cell growth and OME for­ma­tion by flu­o­res­cence light mi­croscopy (fLM), 5. vir­tu­ally si­mul­ta­ne­ous scan the sam­ples by elec­tron cry­oto­mog­ra­phy (ECT) at an­gles rang­ing from –60° to 60° in 1° in­cre­ments (thus cre­at­ing minute to­mo­graphic 'slices'), 6. 3D im­age the to­mo­graphic slices. Here's a guide for you: Fig­ure 2 A shows a to­mo­graphic slice (ECT), 2 B the same level af­ter 3D imag­ing, and Fig­ure 1 the com­plete 3D im­age from the same view­point (you can fol­low this re­con­struc­tion in their Movie S12). By com­bin­ing fLM (steps 4) and ECT (step 5) with a tech­nique dubbed cor­rel­a­tive light-elec­tron mi­croscopy (CLEM) they were able to over­lay pic­tures taken at dif­fer­ent mag­ni­fi­ca­tions and at­tribute par­tic­u­lar OMEs to "their" cells (Fig­ure 3). In the ma­jor­ity of their pic­tures, the OMEs ap­pear as chains of outer mem­brane vesi­cles (OMVs) or, more col­lo­quial, waist‑y mem­brane blebs that are mor­pho­log­i­cally read­ily dis­tin­guished from fla­gella, pili, or ex­tra­cel­lu­lar poly­meric sub­stances, and can ex­ceed the pro­duc­ing cells in length.

But how are these 'fairy lights' switched-on? Which con­stituents of the OMEs are re­spon­si­ble for elec­tron trans­port? S. onei­den­sis pos­sesses a num­ber of periplas­mic and OM-bound mul­ti­heme pro­teins of the cy­tochrome c fam­ily in ad­di­tion to cy­tochromes that re­side in the in­ner mem­brane (IM) that are re­spon­si­ble for elec­tron fun­nel­ing dur­ing res­pi­ra­tion (the hemes are the pros­thetic groups of mul­ti­heme cy­tochrome c pro­teins that trans­port the elec­trons). Of these "non-IM" cyto­chromes, MtrA con­tacts the OM from the periplas­mic side of the vesi­cles, and MtrC sits on the out­side (see here for a di­a­gram). Sub­ra­man­ian et al. ob­served in their elec­tron mi­cro­graphs suspi­cious smaller patches of "knobs" (Fig­ure 2 B,D,E) that cor­re­sponded in size to that of the (known) crys­tal struc­tures of MtrA and MtrC, re­spec­tively. They de­duced from the ob­ser­va­tion that both ty­pes of "knobs" were by >50% re­duced in se­lected OME vesi­cles pro­duced by a Δmtr mu­tant strain that these two pro­teins con­sti­tute part of what makes the OMEs con­duc­tive for elec­trons. Like all mem­brane pro­teins de­void of a cy­to­plas­mic "an­chor" MtrA and MtrC have no fixed po­si­tions in the mem­brane and can lit­er­ally "float" side­ways, and, as they do not cover the mem­branes comple­te­ly but oc­cur in patches, elec­tron trans­port is likely to in­clude elec­tron move­ment by in­ter-mole­cu­lar "hop­ping" in ad­di­tion to in­tra-mol­e­c­u­lar "fun­nel­ing" through stacked hemes.

Fig­ure 3. Tar­get­ing dy­namic OM ex­ten­sions of S. onei­densis  for elec­tron cry­oto­mog­ra­phy (ECT) us­ing correla­tive light and elec­tron mi­croscopy (CLEM). Tar­get lo­cations on fixed and plunge-frozen elec­tron mi­croscopy grids, from the per­fusion flow imag­ing plat­form, were im­aged by ECT, re­veal­ing the OMV chain mor­phol­ogy of the OM ex­ten­sions. A rep­re­sen­ta­tive ECT ima­ge, with cor­responding flu­o­res­cence light micro­scopy (fLM) ima­ge (In­set) (red color: mem­brane stain FM 4–64FX present through­out the expe­ri­ment). ECT scale bars, 100 nm; fLM scale bars, 2 μm. White dot­ted boxes in the fLM im­ages indi­cate the cor­re­spond­ing appro­xi­mate re­gions im­aged in ECT. Source

Cel­lu­lar 'ap­pendages' like the outer mem­brane exten­si­ons (OMEs) of She­wanella are re­ally small and rather frag­ile, es­pe­cially un­der the harsh con­di­tions to which they are ex­posed dur­ing the elec­tron mi­croscopy proce­dure. There­fore, Sub­ra­man­ian et al. took care to not pro­duce ar­ti­facts (some­thing that haunts all elec­tron micro­sco­pists). They mon­i­tored the num­ber and ap­pear­ance of OMEs in un-fixed vs. fixed sam­ples and found that al­though the num­ber of in­tact OMEs was very low in un-fixed sam­ples, they looked in­dis­tin­guish­able from those in glu­taralde­hyde-fixed sam­ples. Also, the con­stric­tions along the OMEs that gives them the ap­pear­ance of chains of waist‑y mem­brane blebs were ob­served un­der both con­di­tions, in­clud­ing the elec­tron-dense ma­te­r­ial (of yet un­known com­po­si­tion) in the periplasm of the blebs (Fi­gure 3). An al­lur­ing but un­proven as­sump­tion is that this in­ter­nal 'belt' is com­posed of re‑purposed com­po­nents of the cel­lu­lar ma­chin­ery nor­mally con­strict­ing cells dur­ing cell di­vi­sion. The re­searchers took time­lapse se­ries of in­di­vid­ual cells to fol­low the pro­tru­sion and re­trac­tion of in­di­vid­ual OMEs and found them to be highly dy­namic: pro­tru­sion and re­trac­tion oc­curred within an hour, ap­prox­i­mately, and in­cluded bud­ding and branch­ing, and po­si­tion­ing and dis­so­lu­tion of the belt-like con­stric­tions. In this re­spect, Shewannella's nanowires re­sem­ble neu­rons with their ex­tend­ing ax­ons more than the cop­per nanowires of the hard­ware store.