Investing in Diversification: Transport of Vitamin B12 Analogs by Microbes of the Human Gut

by Daniel and Elio
 

Both of us have bliss­ful­ly go­ing along in our lives with­out know­ing what cor­ri­noids are, at least not by that name. We had of course learn­ed way back (way, way  back for Elio) that co­ba­la­min (vi­ta­min B₁₂) is a com­pli­ca­ted or­gan­ic mo­le­cu­le made up of pyr­role rings and co­balt, but we hadn't heard of its 'cor­ri­noid' mo­ni­ker. It de­riv­es from cor­rin, a pla­nar te­tra­pyr­role ring (Figure 1). To our know­ledge, Cor­rin – the half-dra­gon pro­ta­gon­ist of Fire Em­blem Fa­tes, a tac­ti­cal role-play­ing vi­deo­game for the Nin­ten­do 3DS – is un­re­la­ted. Cor­ri­noids come in a num­ber of sha­pes and forms, some car­rying co­balt and oth­ers al­ter­na­te me­tals, or even just hy­dro­gen.

Figure 1. Corrin the Nintendo 3DS character in both female and male avatar template foun­da­tions (top), Corrin the molecule (bottom left), and Vitamin B₁₂ (bottom right)

A bit more chemistry. Cor­ri­noids re­sem­ble the por­phy­rin group of a he­me, but dif­fer from it by lack­ing one of the car­bon groups that round up the he­me struc­tu­re. In vi­ta­min B₁₂ the co­balt ion does a lot of work: it co­or­di­nat­es a so-cal­led up­per and low­er axi­al li­gand in ma­ny cor­ri­no­id-de­pen­dent en­zym­es. This works by break­ing the co­balt-car­bon bond. These com­pounds also fa­ci­li­ta­te ra­di­cal-me­di­a­ted re­act­ions, me­thyl group trans­fers, and oth­er che­mi­cal pro­ces­ses.

Daniel discovered in person the im­por­tan­ce of vi­ta­min B₁₂ a few years back when he felt symp­toms of a slight de­fi­cien­cy of it. Vi­ta­min B₁₂ is a co­en­zy­me re­qui­red in people for ma­king mye­lin and red blood cells, plus it is es­sen­tial for al­most all the rest of me­ta­bo­lism. It func­tions as a co­en­zy­me for cer­tain iso­me­ra­ses (whe­re it al­lows the re­ar­ran­ge­ment of hy­dro­gen atoms), me­thyl­trans­fe­ras­es (for the ad­di­tion of me­thyl groups, for ex­am­ple in ma­king me­thio­ni­ne), and de­ha­lo­ge­na­ses (lea­ding to the re­mo­val of a ha­lo­gen atom from an or­gan­ic mo­le­cu­le), plus, plus, plus. In oth­er words, it's a big deal in the me­ta­bo­lism of bacteria, archaea, fungi, ani­mals and some al­gae. Plants, however, do not have B12-dependent enzymes.  Importantly, on­ly bac­te­ria and ar­ch­aea can make it.

Microbiologists would also appre­ci­a­te that vi­ta­min B₁₂ mo­du­lat­es the gut mi­cro­bi­ome. But cor­ri­noids are not just vitamin B₁₂. Au contraire, most bacteria and archaea utilize different forms of the basic mo­le­cu­le. May­be this re­la­tes to the vi­de­o­ga­me cha­rac­ter mo­re than we thought? Like Cor­rin's gen­der, phy­si­cal ap­pear­an­ce, and voice can va­ry from play­er to play­er, so too do the phy­si­cal pro­per­ties of cor­ri­noids used by mi­cro­bi­al spe­cies va­ry. We don't know much about the spe­ci­fi­ci­ty of the va­ri­ous mo­le­cu­lar forms, but keep read­ing the sect­ions be­low to learn a bit. One might sus­pect that the cor­ri­no­id struc­ture would in­flu­en­ce the de­gree to which that par­ti­cu­lar form can used by any giv­en mi­cro­bi­al spe­cies.

Most bacteria (~80% of species) uti­li­ze cor­ri­no­ids, but only a few (~40%) can ma­ke them. Those that do, ma­nu­fac­ture one ty­pe, using bio­syn­the­tic path­ways that can in­vol­ve 30+ ge­nes. Mak­ing one type means that these mo­le­cul­es could be use­ful mar­kers for stu­dy­ing eco­lo­gi­cal re­la­tion­ships. In­deed, cor­ri­no­id me­ta­bo­lism in ge­ne­ral has ser­ved as a novel mo­del to dis­sect prin­cip­les of com­mu­ni­ty struc­ture.

To summarize, although a bacte­ri­al spe­cies can only make one spe­ci­fic cor­ri­no­id (if any), any giv­en spe­cies of­ten has a lar­ge re­per­toire of forms that it can use. How does a giv­en spe­cies spe­cia­lize in its ran­ge of par­ti­cu­lar cor­ri­no­id struc­tur­es?
 

When Redundancy is Economical

Before we answer that question, consider this riddle:

After a baseball player throws a perfect game, what do her teammates get her to celebrate at the bar?
Answer: A pitcher's pitcher of beer, of course.

What does that joke riddle have to do with the topic at hand? Well, re­dun­dan­cy, and cas­es where the same thing can be used for uni­que pur­pos­es – or mean­ings. Func­tio­nal re­dun­dan­cy is a com­mon re­frain in mo­le­cu­lar cell bio­lo­gy and ge­ne­tics: two or more genes, pro­teins, or path­ways car­ry out the same ba­sic job. In ma­ny cas­es this si­tu­a­tion makes sen­se, hav­ing a cel­lu­lar ‘back-up' plan for es­sen­tial life-or-death func­tions in case one play­er is in­ac­ti­vat­ed or goes mis­sing. But in other cas­es the rea­son for this kind of re­dun­dan­cy is not ob­vi­ous, seem­ing in­stead ex­ces­sive and even ener­ge­ti­cal­ly waste­ful.

Figure 2. Redundancies in B₁₂ Transport in the Human Gut Microbiome. (Modified from Deg­nan et al.)

Metagenomic sequencing efforts of the hu­man gut mi­cro­bi­ome has re­veal­ed that most spe­cies ap­pear to en­code mul­tiple vi­ta­min B₁₂ trans­por­ters in their ge­nom­es (Figure 2). Why this re­dun­dan­cy? As you may have al­rea­dy gues­sed, one lo­gi­cal ans­wer is that each pro­te­in has evol­ved to pre­fer dif­fer­ent cor­ri­noid mo­le­cul­es, not just vi­ta­min B₁₂. Just like the com­bi­na­tion of let­ters in 'pit­cher' has evol­ved in Eng­lish to take on two dis­tinct func­tio­nal roles, so too has dup­li­ca­tion of ge­nes al­low­ed slight al­te­ra­tion in their func­tions through evo­lu­tion. The extra ener­gy re­quir­ed for mak­ing two cor­ri­noid trans­port­ers is ba­lanc­ed by the se­cond co­py 'not be­ing com­ple­te­ly the same, just slight­ly dif­fe­rent to trans­port a uni­que cor­ri­noid. The cell now has con­trol over which cor­ri­noid struc­ture it will take up, and each struc­ture li­ke­ly is used with dif­fe­rent ef­fi­cien­cy, uti­li­zed in dif­fe­rent en­vi­ron­ments, or per­haps even used for dif­fe­rent pur­pos­es.

One might even argue that 'func­tio­nal re­dun­dan­cy' is nev­er whol­ly re­dun­dant, but rath­er subtle va­ri­a­tions of a com­mon the­me, the bio­lo­gi­cal equi­va­lents of Bach's Gold­berg Va­ri­a­tions. Ma­ny es­sen­tial genes – such as po­ly­me­ras­es – have no 100% iden­ti­cal func­tio­nal back­ups. Even en­zym­es in bio­che­mi­cal path­ways that may be able to take over for the loss of an­oth­er have an ex­pan­ded re­per­toire of ca­pa­bi­li­ties, not just simp­ly serv­ing as dup­li­ca­te back­up mea­sures.

A paper from a few years back es­tab­lish­ed that there are three B₁₂ trans­por­ter sys­tems en­cod­ed by the pro­mi­nent hu­man gut sym­biont Bacteroides the­ta­io­ta­mi­cron . They ser­ve not me­re­ly in re­dun­dan­cy, but ex­pand the re­per­toire of usable cor­ri­noids for the spe­cies. The auth­ors fur­ther in­ves­ti­gat­ed the con­trol of these cor­ri­no­id trans­port­ers in gno­to­bio­tic mice in va­ri­ous diets of the host, and mea­sur­ed their over­all abun­dan­ce and ef­fects on mi­cro­bi­al fitness.
 

Making the Most of Opportunities

B. the­ta­io­ta­mi­cron  is an ideal mo­del spe­cies for in­ves­ti­ga­ting the po­ten­tial re­dun­dan­cy and dif­fer­ing spe­ci­fi­ci­ties of mul­tiple cor­ri­noid trans­port sys­tems. Whe­re­as E. coli  on­ly has one such sys­tem, B. the­ta­io­ta­mi­cron  has three. It is un­able to syn­the­si­ze its own cor­ri­noids and re­quir­es vi­ta­min B₁₂ as a co­fac­tor for MetH, an en­zy­me in­volv­ed in me­thi­o­ni­ne syn­the­sis. More­over B. the­ta­io­ta­mi­cron  is ge­ne­ti­cal­ly trac­table, un­like some of the other ma­jor com­po­nents of the hu­man mi­cro­bio­ta.

Thus, the authors were able to con­struct de­le­tions in one or more of the three btuB gen­es of the re­dun­dant cor­ri­no­id trans­port sys­tems of B. the­ta­io­ta­mi­cron (more on all the com­po­nents and struc­ture of the­se trans­port sys­tems be­low). The au­thors al­so de­ter­min­ed which mu­tant strains were able to sur­vi­ve and grow in me­dia con­tain­ing vi­ta­min B₁₂ but lack­ing me­thi­o­ni­ne. All the sing­le and double mu­tants were able to grow on their own, but the triple mu­tant was not, in­di­ca­ting that the­se cor­ri­noid trans­port sys­tems are in­deed re­dun­dant. Any sing­le trans­por­ter is suf­fi­cient for im­port­ing vi­ta­min B₁₂ on its own, but hav­ing at least one func­tio­nal sys­tem is es­sen­tial.

The authors then quanti­fied the con­tri­bu­tion of each BtuB ho­mo­log (BtuB1 – 3) in com­pe­ti­ti­ve growth as­says of mix­ed wild type and ΔbtuB mu­tants in me­dia con­tain­ing vi­ta­min B₁₂ but lack­ing me­thi­o­ni­ne. The re­sults (Figure 3A) show­ed that the strain en­co­ding BtuB1 is se­ve­re­ly out­com­pe­ted by wild type cells, the strain with on­ly BtuB2 com­pe­tes equi­va­lent­ly with wild type cells, and the one with only BtuB3 has an in­ter­me­di­a­te phe­no­ty­pe. The­re­fo­re, al­though re­dun­dant, the three cor­ri­noid trans­port sys­tems show dif­fer­ing abi­li­ties for trans­port­ing vi­ta­min B₁₂.

(Click to enlarge )
Figure 3. Functional Specificity for Redundant Corrinoid Transporters of B. the­ta­io­ta­mi­cron . (from Degnan et al.). A Com­pe­ti­tion assays between indicated strains. Wild ty­pe, mutant, and complemented strains were in­ocu­lated at an initial ratio of 1:8:1 and passaged daily for five days. B Direct competition between strains encoding only BtuB1 or BtuB3 in the pre­sence of variable corrinoid structures. C Mole­cu­lar structure of vitamin B₁₂ and the lower li­gands of the other corrinoids examined. Dash­ed lines outline the precursor cobinamide, lea­ving the variable lower ligand portion outside the dashes.

They then used seven corri­noid va­ri­ants to test the re­la­ti­ve fit­ness of the ΔbtuB mu­tants in their pre­sen­ce (Figure 3B). These se­ven struc­tur­es re­pre­sent on­ly a small frac­tion of known cor­ri­noids pre­sent in the hu­man gut from diet and mi­cro­bi­al syn­the­sis. How­ev­er, they do span across the three known struc­tu­ral fa­mi­lies (Figure 3C). As can be seen in Fi­gure 3B, the re­la­ti­ve fit­ness of strains en­co­ding single BtuB ho­mo­logs did dif­fer ba­sed upon which cor­ri­noid struc­ture was pre­sent, ar­gu­ing that the three sys­tems are not just re­dun­dant, but al­so re­pre­sent the abi­li­ty of B. the­ta­io­ta­mi­cron  to make the most of eve­ry en­vi­ron­men­tal op­por­tu­ni­ty that pre­sents it­self. One can ima­gi­ne that hav­ing the three func­tio­nal sys­tems would give the spe­cies com­pe­ti­ti­ve ad­van­tage with­in the gut re­gard­less of which cor­ri­no­id struc­ture is most abun­dant.

Indeed, moving from growth in culture me­di­um to the gno­to­bi­o­tic mouse, the au­thors re­port­ed a ~4,000-fold re­duct­ion in the com­pe­ti­ti­ve fit­ness of the strain lack­ing only BtuB2. The role of BtuB2 in spe­ci­fi­cal­ly im­port­ing vi­ta­min B₁₂ was high­light­ed by its even great­er im­por­tan­ce for B. the­ta­io­ta­mi­cron  sur­vi­val in mice that were de­ple­ted of the vi­ta­min in their diet. As in the ex­pe­ri­ments in cul­ture, strains en­co­ding BtuB3 out­com­pe­ted strains en­co­ding Btub1 only, with­in the mouse gut.
 

How Varied is Corrinoid Transporter Specificity?

By looking at genomic data of known human gut microbes, the authors could cluster BtuB ho­mo­logs from over 300 species. This analysis conservatively predicts that 27 corrinoid transporter fa­milies share less than 50% protein sequence identity with each other. One could therefore predict that these 27 families should have distinct corrinoid specificities.

A question you may have been, or are now, asking: Which component of the corrinoid transport system is BtuB, and why is this the gene/protein that the authors chose as the basis for their stu­dy? Corrinoid transporters fall in the ATP-binding cassette (ABC) transporter system family. BtuCD is the ABC component of this system, spanning the cellular membrane. It is conserved throughout Bacteria. BtuF, a binding (chaperone) component, is also well conserved (it is extracellular for Gram-positives, or in the periplasm of Gram-negatives). Finally, BtuB is dependent on the TonB outer-membrane transport component, and is of course only found in Gram-negative species. Many, but not all of these transport systems are regulated by a riboswitch. The authors' estimate of the number of coronoid transporters is conservative mainly because their focus on the BtuB component leaves out any possible Gram-positive transport systems (which are poorly known). The Firmicutes are major components of the human microbiota and at least half encode possible corrinoid transport systems. Additionally, it is likely that many other important microbial species present within the human gut have not yet been sequenced.

So, why choose BtuB as the element of study and comparison for corrinoid transport systems if it is only present in Gram-negative bacteria? The reason is that using any of the other system com­po­nents as a basis for identification and comparison would create far too many false positives. BtuB seems specific for corrinoids, whereas the other components are involved in the transport of other substrates as well.

Additionally the authors found that any the BtuFCD components present in B. the­ta­io­ta­mi­cron  (that is to say BtuFCD1 – 3 from any of the three redundant systems encoded by the species) could perhaps interact with BtuB1, 2, or 3. This observation raises the possibility that additional spe­ci­fi­ci­ty for a particular corrinoid could arise through mixing among multiple-encoded systems within a cell, for instance to form a transporter system made up of BtuB1 with BtuF2 and BtuCD3. If such mixing of redundant systems within a cell occurs, that then makes the authors' estimates of the total number of distinct carrinoid spe­ci­fi­ci­ties within the gut even more conservative.
 

Investing in Diversification

We now come full circle to our discovery of the term corrinoid and how corrinoids are produced and used by bacteria. We are left with many unknowns. Why do species limit themselves to pro­duc­tion of just one corrinoid structure, but utilize multiple ones? Are these structures ways of com­mu­ni­ca­ting 'self' or ‘other' to one another within a community? The authors of the paper we've focus­ed on speculate that these small molecules may be involved in community sensing and responses.

From this study, it does seem likely that the evolution of multiple corrinoid transport system al­lows both some measure of redundancy and also expands a species' repertoire of substrates that can be captured for a common function. Such an expanded repertoire may allow the species to survive and outcompete others within a community faced with environmental fluctuations, such as the changing level of metabolites in the mammalian gut. It also could conceivably lead to com­ple­tely new uses for distinct substrate in diverse processes. Spending the extra energy involved to invest in such diversification may be well worth it.