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.
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.
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.
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.