Through the Look­ing Glass: Sil­i­cate in Bac­te­r­ial Spores

by Pe­ter Set­low

Amid the furor sur­round­ing the an­thrax at­tacks in the USA in 2001, sig­nif­i­cant at­ten­tion was fo­cused on whe­ther the Bacil­lus an­thracis spores used had been "wea­ponized" by the adding of a coat­ing of fumed sil­ica to aid in their dis­per­sal. This in­for­ma­tion might have helped pin­point where the spores had orig­i­nated. How­ever, a con­fus­ing fac­tor was that for more than 20 years, signi­ficant lev­els of sil­i­con had been re­ported in spores of at least some Bacil­lus species, in­clud­ing those of Bacil­lus cereus, a close rel­a­tive of B. an­thracis. Clearly, the natu­ral pres­ence of sil­i­con in B. an­thracis spores makes dis­crimination be­tween weaponized and non-weaponized spores more dif­fi­cult. How­ever, in the older re­ports the sil­i­con was not lo­cal­ized at the out­er­most sur­face of the spores, as would be ex­pected if they had been ar­ti­fi­cially spiked with such com­pounds.

Sil­i­con lo­ca­tion in spores. Spores of a high sil­i­cate-ad­sorb­ing Bacil­lus iso­late of the B. cereus group were pre­pared in si­li­cate-con­tain­ing medium. TEM Panel: an ul­tra­thin sec­tion of a spore by trans­mis­sion elec­tron mi­croscopy. The gran­ules on the outer sur­face of the coat were ab­sent from spores pre­pared in sil­i­cate-free medium. (CX = pep­ti­do­gly­can cor­tex; UC = un­der­coat; CT = coat; SX = sil­i­con-con­­tain­ing gran­ules; EX = out­er­most exo­sporium layer.) SI‑K Panel: STEM-EDX im­age with the sil­i­con pseudo-col­ored green. Merge Panel: TEM and Si‑K im­ages com­bined. Bar = 100 nm. Source

Any­how, what is sil­i­con do­ing on a bac­te­r­ial spore? A re­cent pa­per pro­vides some clues. Start­ing with soil from a paddy field, these re­searchers iden­ti­fied a num­ber of novel bac­te­r­ial iso­lates that ef­fi­ciently take up sil­i­cate present in me­dia at con­cen­tra­tions com­pa­ra­ble to lev­els found in the wa­ter in soils. Strik­ingly, all of these iso­lates were Bacil­lus species and most were mem­bers of the B. cereus group that in­cludes the pathogens B. an­thracis, B. cereus, and Bacil­lus thuringien­sis. Sil­i­cate up­take by the novel iso­lates was as­so­ci­ated only with spore for­ma­tion (sporu­la­tion), and was a late event in this process. How­ever, while sporu­la­tion was re­quired for ef­fi­cient sil­i­cate up­take, sil­i­cate up­take it­self was not es­sen­tial for sporu­la­tion.

Vi­su­al­iz­ing sil­i­cate on the spores of these new iso­lates re­quired some fancy tech­niques, namely scan­ning elec­tron mi­croscopy cou­pled with en­ergy dis­per­sive X‑ray spectro­metry (STEM-EDX). The sil­i­cate taken up was present, at least in part, as gran­ules on the outer sur­face of the spore coat, with some per­haps in the coat it­self. Note that the out­er­most layer in B. an­thracis and B. cereus spores is not the coat but rather the ex­ospo­rium, a loosely-fit­ting out­er­most struc­ture found on some, but not all, bac­te­r­ial en­dospores. Analy­sis of sil­i­cate up­take showed that labora­tory B. cereus and Bacil­lus thuringien­sis strains, as well as a more dis­tantly re­lated Bacil­lus mega­te­rium strain, also ac­cu­mu­lated sil­i­cate on spores, al­though at lower lev­els than the novel Bacil­lus iso­lates. In­ter­est­ingly, the well-char­ac­ter­ized Bacil­lus sub­tilis 168 lab­o­ra­tory strain took up no de­tectable sil­i­cate. These new re­sults make it clear that spores of at least some Bacil­lus species in­cor­po­rate sig­nif­i­cant amounts of sil­i­cate and de­posit it on the spore coat. It is no­table that this sil­i­cate is not on the spore's outer layer, and thus it should be pos­si­ble to dis­crim­i­nate be­tween weaponized and non-weaponized B. an­thracis spores by STEM-EDX analy­sis.

Bioter­ror­ism aside, this work raises sev­eral in­ter­est­ing ques­tions. The first con­cerns the mecha­nism of sil­i­cate ac­cu­mu­la­tion on spores. It ap­pears to be me­di­ated by the mother cell in which the spore ma­tures, but how the mother cell does this is not known. Find­ing this out may lead to an un­der­stand­ing of why some Bacil­lus strains/species ac­cu­mu­late sil­i­cate and some do not. The sec­ond ob­vi­ous ques­tion is why would a spore ac­cu­mu­late sil­i­cate at lev­els up to 6% of the spore's dry weight. One pos­si­bil­ity is that this might fa­cil­i­tate dis­per­sion of sin­gle spores, much as does an outer coat­ing of fumed sil­ica. How­ever, ac­cord­ing to this pa­per, high lev­els of sil­i­cate on the spore coat did not en­hance the dis­per­sal of dry spores. Thus, sil­i­cate ac­cu­mu­la­tion must play an­other role.

In­deed, whereas the spores' sil­i­cate plays no role in spore re­sis­tance to heat, hy­dro­gen per­ox­ide, UV ra­di­a­tion or NaOH, it sig­nif­i­cantly in­creases spore re­sis­tance to killing by 0.1–0.4 N min­eral acids. This in­creased acid re­sis­tance might be par­tic­u­larly im­por­tant in spores of pathogens such as B. cereus and B. an­thracis that may pass through an acidic mam­malian di­ges­tive tract. On the other hand, this would not be im­por­tant in the al­ka­line di­ges­tive tract of the in­sect forms for which B. thuringien­sis is path­o­genic. There­fore, it seems likely that the spores' sil­i­cate layer may serve an ad­di­tional func­tion. Since sil­i­cate ac­cu­mu­la­tion in other or­gan­isms can im­part struc­tural rigid­ity, per­haps sil­i­cate plays such a role for spores as well. This leaves us with yet more inter­esting ques­tions to ad­dress to these spores.

Pe­ter Set­low is Pro­fes­sor of Mol­e­c­u­lar, Mi­cro­bial and Struc­tural Bi­ol­ogy at the Uni­ver­sity of Con­necti­cut Health Cen­ter in Farm­ing­ton, CT.