Tales of Mys­tery and Imag­i­na­tion (1/2)

by Daniel P. Haeusser

The Story so far

Over the last ten-plus years of this blog's ex­is­tence, one clus­ter of branches on the bac­te­r­ial phy­lo­ge­ne­tic tree in par­tic­u­lar has pre­sented us with fas­ci­nat­ing mys­ter­ies re­gard­ing ba­sic, and seem­ing­ly unique, cel­lu­lar bi­ol­ogy. Many as­pects of the species on these branches re­main enig­matic, but what de­tails re­searchers have been able to coax from them con­tinue to in­duce sharp turns in our mi­cro­bial as­sump­tions and un­der­stand­ing: con­cep­tual an­gles that mimic the bumpy, un­even cel­lu­lar sur­face of some of those species un­der dis­cus­sion (Fig­ure 1).

Fig­ure 1. Mem­bers of the Planc­to­my­ce­tes–Ver­ru­co­mi­cro­bia–Chla­my­diae super­phylum (PVC) arose from a com­mon Gram-neg­a­tive an­ces­tor and presently dis­play vari­a­tions in cell shape and cy­toki­ne­sis. Like present-day E. coli, the Gram-neg­a­tive an­ces­tor con­tained a pe­ri­plas­mic pep­ti­do­gly­can cell wall (dashes), em­ployed an MreB cy­toskele­ton (not shown) to elon­gate, and used an FtsZ cy­toskele­ton to con­strict (mid­cell black dot). Re­cent ev­i­dence in­di­cates that Planc­to­mycetes have re­tained pep­ti­do­gly­can cell walls. Some mem­bers have also re­tained FtsZ-based cy­toki­ne­sis (not shown), but many have lost FtsZ and now di­vide by bud­ding, even with cell walls. The Ver­ru­comi­cro­bia have kept cell wall and di­vi­sion com­po­nents of their evo­lu­tio­nary an­ces­tor, but of­ten have outer mem­brane 'wart-like' ex­ten­sions that are not well char­ac­ter­ized. Some Chlamy­diae have re­tained cell wall all along their length (not shown), but path­o­genic species have very low amounts of pep­tido­glycan syn­the­sis, lim­ited to the site of di­vision dur­ing cy­toki­ne­sis. Ad­di­tion­ally for un­known rea­sons, their cy­toki­ne­sis has evolved to oc­cur slightly asym­met­ri­cally (ex­ag­ger­ated in the il­lus­tra­tion for em­pha­sis). The re­cent re­ve­la­tions of pep­ti­do­gly­can be­ing present through­­out the PVC su­per­phy­lum leave the Mol­li­cutes (il­lus­trated here with the rep­re­sen­ta­tive My­co­plas­ma mo­bile ) as the only main group of bac­te­ria that seem to have evolved to lack pep­ti­do­gly­can and to have also lost FtsZ (present in their Gram-pos­i­tive an­ces­tor). De­spite this loss, Mol­li­cute species like M. mo­bile have com­plex shapes and un­char­ac­ter­ized cy­toskele­tons. I­llu­stration by Allyson Corigliano (Can­i­sius Col­lege) based on the sum­mary fig­ure of the Fron­tiers in Mi­cro­bi­ol­ogy re­view source.

That clus­ter of branches (if you haven't twigged it yet) would be the Planctomycetes–Verrucomicrobia–Chla­my­di­ae su­per­phy­lum (PVC). If you fondly re­call our past vi­sits with these mi­crobes, you could skip on to the next sec­tion. But for those want­ing a lit­tle con­text be­fore delv­ing into new de­vel­op­ments on the PVC front, a re­cap and in­tro­duc­tion:

Though men­tioned on oc­ca­sion, we haven't re­ally fea­tur­ed any­thing on the Chy­lamy­diae branch, a phy­lum of ob­ligate in­tra­cel­lu­lar bac­te­ria that in­cludes, but is not li­mit­ed to, the well-known path­o­genic Chlamy­dia fam­ily. As is com­mon for ob­lig­ate in­tra­cel­lu­lar or­gan­isms, these bac­te­ria have re­duced genomes (which will have im­por­tance dis­cussed fur­ther be­low). But they are par­tic­u­larly no­table for their cel­lu­lar de­vel­op­ment into an el­e­men­tary body, a non-repli­cat­ing form in the nanome­ter-di­am­e­ter range that can trans­mit be­tween host cells via the ex­tra­cel­lu­lar mi­lieu. Though of­ten com­pared to a spore, it might be more ap­pro­pri­ate to view the el­e­men­tary body as a 'virion' mode of be­hav­ior for the Chlamy­diae phy­lum species.

One of my first posts for this blog fea­tured the dis­cov­ery of tubu­lins in a Pros­the­cobac­ter mem­ber of the Ver­ru­co­mi­cro­bia, a phy­lum that Elio men­tioned in a 'whiff of ta­xo­no­my' post that spec­u­lated on their epony­mously war­ty ap­pear­ance. To my knowl­edge, the func­tions of the pros­the­cae and the warty bumps of the var­i­ous mem­bers of this phy­lum are still un­ver­i­fied, though in the­ory they might be ex­pected to func­tion in sur­face ad­he­sion, as in the pros­th­e­cate Al­phapro­teobac­te­ria, and/or to pro­vide a greater mem­brane sur­face area.

A decade ago, a group of grad stu­dents wrote on the "far out" Planc­to­mycetes, a phy­lum known for tan­ta­liz­ingly eu­kary­otic-like fea­tures, such as ex­ten­sive en­do­mem­brane for­ma­tion, in­clud­ing the ap­par­ently mem­brane– bound bac­te­r­ial or­ganelle hous­ing the com­plex res­pon­sible for anaer­o­bic ox­i­da­tion of am­mo­nium. In­ter­est in the Planc­to­mycetes par­tic­u­larly surged with the dis­co­ve­ry of mem­brane com­part­men­tal­iza­tion around DNA in Gem­mata ob­scuriglobus. How­ever, as cov­ered here by an­other grad­u­ate stu­dent con­trib­u­tor, this pur­ported prokary­otic 'nu­cleus' seems to be formed by a bi­layer con­tin­u­ous with, not in­de­pen­dent of, the cell mem­brane. The last word is not in on this topic, as the pur­ported 'nu­clear mem­brane' con­tains nu­clear-pore-like struc­tures.

An Aside on Planc­to­mycete Chro­mo­some Repli­ca­tion

What can you do if you don't want to wait pa­tiently for more de­tailed stud­ies of the enig­matic mem­brane com­part­men­tal­iza­tion around the nu­cleoid in Gem­mata ob­scuriglobus ? Ask a co-blog­ger ! My col­league Christoph had al­ready con­tributed in­sights from bioin­for­mat­ics to chro­mo­some re­plication of the CPR bac­te­ria. So here is what he said.

Fig­ure 2. A Schematic re­pre­sen­ta­tion of pre­dicted oriC struc­tures for 5 Planc­to­mycetes. Red half­-cir­cles: pre­dicted DnaA-bind­ing sites; or­ange el­lipses: pre­dicted DNA-un­wind­ing el­e­ments (DUE); blue el­lipses: DnaA ss­DNA-bind­ing mo­tif (DnaA-trio); gray boxes: flank­ing genes. The oriCs of B. sub­tilis and E. coli are shown for com­par­i­son. C. Weigel, un­pub­lished. B Do­main struc­ture of the E. coli DnaA pro­tein. Source

The 15 genomes of the Planc­to­mycetes that have been se­quenced so far are rather large (3 – 10 Mb, in most cases ap­prox­i­mately twice the size of the E. coli genome) and they are all sin­gle cir­cu­lar chro­mo­somes. For five of them, in­clud­ing G. ob­scuriglobus, the repli­ca­tion ori­gin oriC can be safely pre­dicted to lo­cal­ize in the ~300 – 500 bp long in­ter­genic re­gion be­tween the dnaA‑2 and dnaN genes (Fig­ure 2A). This ge­nomic lo­ca­tion for oriC is quite com­mon for bac­te­ria from all known phyla but this is not a strict rule. The best-stud­ied model, E. coli, be­ing a pro­minent ex­cep­tion. The oriC struc­ture of these five Planc­to­my­ce­tes is fairly sim­i­lar to the known repli­ca­tion ori­gins of B. sub­tilis and E. coli (shown in Fig­ure 2A for an eas­ier com­par­i­son). They con­tain a promi­nent DNA-un­wind­ing el­e­ment (DUE), that is, a short stretch of ~50 bp that be­comes eas­ily sin­gle-stranded when neg­a­tively su­per­coiled. Also, they all con­tain a 'DnaA-trio' mo­tif flank­ing the DUE, which has re­cently been shown to bind DnaA to sin­gle-stranded DNA dur­ing the ini­ti­a­tion step of chro­mo­some repli­ca­tion in B. sub­tilis. Three of the Planc­to­mycetes oriCs lack a hall­mark of all known oriCs: a DnaA‑binding site in re­verse ori­en­ta­tion flank­ing the DUE (marked 'R1_{E. coli'} in Fig­ure 2A). This R1 DnaA‑binding site is thought to be im­por­tant for load­ing of the he­li­case dur­ing ini­ti­a­tion of chro­mo­some repli­ca­tion, and the mech­a­nism might there­fore be dif­fer­ent in the Planc­to­mycetes. This lat­ter sus­pi­cion is re­in­forced by the ob­ser­va­tion that the Planc­to­my­ce­tes genomes con­tain two dnaA genes (en­cod­ing the repli­ca­tion ini­tia­tor pro­tein), and one of these genes, dnaA‑2, lacks the N‑terminal do­main 1 known to be im­por­tant for in­ter­ac­tion with the he­li­case, while the dnaA‑1 genes en­code full-length DnaA or­thologs (Fig­ure 2B). Note that two dnaA genes are very rarely found in bac­te­r­ial genomes, the Chlamy­diae and one branch of the Del­ta­pro­teo­bac­teria, the Desul­fovib­ri­onales, be­ing ex­cep­tions but in both cases the twin dnaA genes are full-length or­thologs. So, taken to­gether, it is safe to pre­dict that chro­mo­some repli­ca­tion in the Planc­to­mycetes is not all that dif­fer­ent from other bac­te­ria for the ini­ti­a­tion step, but the he­li­case-load­ing step cer­tainly re­quires more de­tailed analy­ses.

The chro­mo­some seg­re­ga­tion ma­chin­ery in the Planc­to­mycetes shows com­mon fea­tures in that all genomes en­code or­thologs of the ParA and ParB pro­teins, and the ParABS sys­tem is there­fore prob­a­bly op­er­a­tive. Also, all Planc­to­mycetes en­code or­thologs of the FtsK DNA translo­case. How­ever, the Planc­to­mycetes Ft­sKs lack the N‑terminal mem­brane-an­chor­ing do­main of E. coli FtsK and also the dis­tri­b­u­tion of KOPS mo­tifs (=FtsK-bind­ing sites) is not skewed (=gra­di­ent from oriC to the ter­mi­nus) as in the E. coli genome but more ran­dom, for ex­am­ple, in the genome of Pirellula sta­leyi DSM 6068. This in­di­cates that, to­gether with the lack of FtsZ in the Planc­to­mycetes (see above), the late steps of chro­mo­some seg­re­ga­tion and sub­se­quent cell di­vi­sion are cer­tainly quite dif­fer­ent from your gar­den-va­ri­ety rod-shaped bac­te­ria like B. sub­tilis and E. coli.

So then, aside from hav­ing odd shapes or con­tain­ing eu­kary­otic-like fac­tors and en­domem­brane struc­tures, what makes the PVC su­per­phy­lum so uniquely bizarre for bac­te­ria?

Di­vi­sion and Growth Non­con­formists?

With a few ex­cep­tions, bac­te­ria con­tain cell walls, pre­dom­i­nantly com­posed of pep­ti­do­gly­can. Most char­ac­ter­ized bac­te­ria di­vide by bi­nary fis­sion, and even among those that use other mech­a­nisms, most still uti­lize the prokary­otic tubu­lin FtsZ in the cy­to­ki­netic process. Among the bac­te­r­ial non­con­formists who seemed to abide with­out cell wall and/or di­vide in­de­pen­dently of FtsZ, many seemed to fall within the PVC su­per­phy­lum. A re­cent re­view in Fron­tiers in Mi­cro­bi­ol­ogy presents an evo­lu­tion­ary per­spec­tive on some long-stand­ing mys­ter­ies re­gard­ing PVC su­per­phy­lum cell di­vision and the his­toric lack of ob­served cell wall syn­the­sis in PVC phyla.

It is not par­tic­u­larly sur­pris­ing to see the two non­con­for­mi­ties (in cell wall and in cell di­vi­sion) go hand-in-hand. If a bac­te­r­ial species has evolved to have a cell wall, it is nec­es­sary to pro­duce and turnover that struc­ture dur­ing growth, in­clud­ing for the growth that must ac­com­pany cy­toki­ne­sis. In­deed, for some bac­te­ria, di­vi­sional growth is their only means of growth. The ma­jor roles of FtsZ are in pro­vid­ing a force for mem­brane con­stric­tion and a foun­da­tion for the lo­cal­iza­tion and ac­ti­vity of cell wall syn­the­sis en­zymes, as two re­cent pa­pers (here and here) demon­strate. Thus, were a species no longer re­quire pep­ti­do­gly­can struc­ture for its fit­ness, its di­vi­sion ma­chi­ne­ry could be the­o­ret­i­cally be­come FtsZ in­de­pen­dent. That is, if an­other means of mem­brane con­striction were avail­able. Con­versely, were a species to de­velop an al­ter­nate means of di­vi­sion and lose ftsZ, it would re­quire some other fac­tor for guid­ing wall syn­the­sis dur­ing di­vi­sion, or be freed to lose that cell wall struc­ture.

For the non­con­formist mem­bers of the PVC su­per­phy­lum, how did they evolve their al­ter­nate di­vision strate­gies or evolve the abil­ity to do away with pep­ti­do­gly­can struc­ture? And how much are the two linked? Be­fore an­swer­ing that ques­tion, how­ever, we have to ques­tion the un­der­ly­ing as­sumption that sev­eral PVC su­per­phy­lum mem­bers lack pep­ti­do­gly­can. This as­sump­tion hasn't been based so much on sci­en­tific ob­ser­va­tion, as much as on the his­tor­i­cal lack of sci­en­tific ob­ser­va­tion of pep­ti­do­gly­can cell wall struc­ture in cer­tain PVC su­per­phy­lum species. Re­cent data now in­di­cates that more PVC mem­bers con­tain cell wall than as­sumed. So stay tuned for a sec­ond part of this post! I will dis­cuss then what the past un­derstanding of cell wall con­ser­va­tion in bac­te­ria was, and how it has changed...

Fron­tispiece: Cryo-elec­tron to­mog­ra­phy micro­graph and re­con­structed, av­er­aged to­mo­graph­ic slice sub­frames of Planc­to­my­ces lim­no­philus cells. Source