Phylogenetic distribution and experimental characterization of corrinoid production and dependence in soil bacterial isolates.

Soil microbial communities impact carbon sequestration and release, biogeochemical cycling, and agricultural yields. These global effects rely on metabolic interactions that modulate community composition and function. However, the physicochemical and taxonomic complexity of soil and the scarcity of available isolates for phenotypic testing are significant barriers to studying soil microbial interactions. Corrinoids-the vitamin B12 family of cofactors-are critical for microbial metabolism, yet they are synthesized by only a subset of microbiome members. Here, we evaluated corrinoid production and dependence in soil bacteria as a model to investigate the ecological roles of microorganisms involved in metabolic interactions. We isolated and characterized a taxonomically diverse collection of 161 soil bacteria from a single study site. Most corrinoid-dependent bacteria in the collection prefer B12 over other corrinoids, while all tested producers synthesize B12, indicating metabolic compatibility between producers and dependents in the collection. Furthermore, a subset of producers release B12 at levels sufficient to support dependent isolates in laboratory culture at estimated ratios of up to 1000 dependents per producer. Within our isolate collection, we did not find strong phylogenetic patterns in corrinoid production or dependence. Upon investigating trends in the phylogenetic dispersion of corrinoid metabolism categories across sequenced bacteria from various environments, we found that these traits are conserved in 47 out of 85 genera. Together, these phenotypic and genomic results provide evidence for corrinoid-based metabolic interactions among bacteria and provide a framework for the study of nutrient-sharing ecological interactions in microbial communities.

Soil microbial community response to corrinoids is shaped by a natural reservoir of vitamin B12.

Soil microbial communities perform critical ecosystem services through the collective metabolic activities of numerous individual organisms. Most microbes use corrinoids, a structurally diverse family of cofactors related to vitamin B12. Corrinoid structure influences the growth of individual microbes, yet how these growth responses scale to the community level remains unknown. Analysis of metagenome-assembled genomes suggests corrinoids are supplied to the community by members of the archaeal and bacterial phyla Thermoproteota, Actinobacteria, and Proteobacteria. Corrinoids were found largely adhered to the soil matrix in a grassland soil, at levels exceeding those required by cultured bacteria. Enrichment cultures and soil microcosms seeded with different corrinoids showed distinct shifts in bacterial community composition, supporting the hypothesis that corrinoid structure can shape communities. Environmental context influenced both community and taxon-specific responses to specific corrinoids. These results implicate corrinoids as key determinants of soil microbiome structure and suggest that environmental micronutrient reservoirs promote community stability.

Laboratory evolution of E. coli with a natural vitamin B12 analog reveals roles for cobamide uptake and adenosylation in methionine synthase-dependent growth.

The majority of bacteria use cobamides as cofactors for methionine synthesis or other diverse metabolic processes. Cobamides are a structurally diverse family of cofactors related to vitamin B12 (cobalamin), and most bacteria studied to date grow most robustly with particular cobamides. Because different environments contain varying abundances of distinct cobamides, bacteria are likely to encounter cobamides that do not function efficiently for their metabolism. Here, we performed a laboratory evolution of a cobamide-dependent strain of Escherichia coli with pseudocobalamin (pCbl), a cobamide that E. coli uses less effectively than cobalamin for MetH-dependent methionine synthesis, to identify genetic adaptations that lead to improved growth with less-preferred cobamides. After propagating and sequencing nine independent lines and validating the results by constructing targeted mutations, we found that increasing expression of the outer membrane cobamide transporter BtuB is beneficial during growth under cobamide-limiting conditions. Unexpectedly, we also found that overexpression of the cobamide adenosyltransferase BtuR confers a specific growth advantage in pCbl. Characterization of this phenotype revealed that BtuR and adenosylated cobamides contribute to optimal MetH-dependent growth. Together, these findings improve our understanding of how bacteria expand their cobamide-dependent metabolic potential.

Cobalamin Riboswitches Are Broadly Sensitive to Corrinoid Cofactors to Enable an Efficient Gene Regulatory Strategy.

In bacteria, many essential metabolic processes are controlled by riboswitches, gene regulatory RNAs that directly bind and detect metabolites. Highly specific effector binding enables riboswitches to respond to a single biologically relevant metabolite. Cobalamin riboswitches are a potential exception because over a dozen chemically similar but functionally distinct cobalamin variants (corrinoid cofactors) exist in nature. Here, we measured cobalamin riboswitch activity in vivo using a Bacillus subtilis fluorescent reporter system and found, among 38 tested riboswitches, a subset responded to corrinoids promiscuously, while others were semiselective. Analyses of chimeric riboswitches and structural models indicate, unlike other riboswitch classes, cobalamin riboswitches indirectly differentiate among corrinoids by sensing differences in their structural conformation. This regulatory strategy aligns riboswitch-corrinoid specificity with cellular corrinoid requirements in a B. subtilis model. Thus, bacteria can employ broadly sensitive riboswitches to cope with the chemical diversity of essential metabolites. IMPORTANCE Some bacterial mRNAs contain a region called a riboswitch which controls gene expression by binding to a metabolite in the cell. Typically, riboswitches sense and respond to a limited range of cellular metabolites, often just one type. In this work, we found the cobalamin (vitamin B12) riboswitch class is an exception, capable of sensing and responding to multiple variants of B12-collectively called corrinoids. We found cobalamin riboswitches vary in corrinoid specificity with some riboswitches responding to each of the corrinoids we tested, while others responding only to a subset of corrinoids. Our results suggest the latter class of riboswitches sense intrinsic conformational differences among corrinoids in order to support the corrinoid-specific needs of the cell. These findings provide insight into how bacteria sense and respond to an exceptionally diverse, often essential set of enzyme cofactors.

Purification and detection of vitamin B12 analogs.

Cobamides are a family of enzyme cofactors that are required by organisms in all domains of life. Over a dozen cobamides exist in nature although only cobalamin (vitamin B12), the cobamide required by humans, has been studied extensively. Cobamides are exclusively produced by a subset of prokaryotes. Importantly, the bacteria and archaea that synthesize cobamides de novo typically produce a single type of cobamide, and furthermore, organisms that use cobamides are selective for certain cobamides. Therefore, a detailed understanding of the cobamide-dependent metabolism of an organism or microbial community of interest requires experiments performed with a variety of cobamides. A notable challenge is that cobalamin is the only cobamide that is commercially available at present. In this chapter, we describe methods to extract, purify, and quantify various cobamides from bacteria for use in laboratory experiments.

Risks and benefits of concurrent treatment with antiplatelet and anticoagulation therapy in post-op bioprosthetic aortic valve replacement patients.

BACKGROUND AND AIM: Among patients receiving surgical bioprosthetic aortic valve replacement (bAVR), there is an elevated risk of thromboembolic events postoperatively. However, the risks and benefits of varying anticoagulation strategies remain controversial. The aim of this study is to compare the risks and benefits of aspirin monotherapy to aspirin plus warfarin ("concurrent therapy") in patients receiving bAVR. METHODS: A retrospective cohort study was conducted using patients' data from Kaiser Permanente Northern California, including those who underwent bAVR with or without coronary artery bypass grafting between 2009 and 2018. Patients were identified as having been discharged with aspirin only or concurrent therapy. The outcomes were mortality, thromboembolic events, and clinically relevant bleeding during a 6-month follow-up. The event rates were compared using the Kaplan-Meier method. Multivariable survival analysis, incorporating propensity scores, was used to estimate adjusted hazard ratios (aHRs) for each outcome. RESULTS: The cohort consisted of 3047 patients. Approximately 58% of patients received aspirin only and 42% received concurrent therapy. Patients who received concurrent therapy were more likely to be older, have hypertension, previous stroke, and longer hospital stays. After adjustment using multivariable analysis, concurrent therapy was associated with a higher risk of clinically relevant bleeding (aHR, 2.33; 95% confidence interval, 1.67-3.25). There was no significant difference in the risk of thromboembolic events or mortality between the two groups. CONCLUSION: Patients who underwent bAVR and were discharged on concurrent therapy compared to aspirin only had a significantly increased risk of bleeding without a significant difference in thromboembolic events.

Identification of a Novel Cobamide Remodeling Enzyme in the Beneficial Human Gut Bacterium Akkermansia muciniphila.

The beneficial human gut bacterium Akkermansia muciniphila provides metabolites to other members of the gut microbiota by breaking down host mucin, but most of its other metabolic functions have not been investigated. A. muciniphila strain MucT is known to use cobamides, the vitamin B12 family of cofactors with structural diversity in the lower ligand. However, A. muciniphila MucT is unable to synthesize cobamides de novo, and the specific forms that can be used by A. muciniphila have not been examined. We found that the levels of growth of A. muciniphila MucT were nearly identical with each of seven cobamides tested, in contrast to nearly all bacteria that had been studied previously. Unexpectedly, this promiscuity is due to cobamide remodeling-the removal and replacement of the lower ligand-despite the absence of the canonical remodeling enzyme CbiZ in A. muciniphila We identified a novel enzyme, CbiR, that is capable of initiating the remodeling process by hydrolyzing the phosphoribosyl bond in the nucleotide loop of cobamides. CbiR does not share similarity with other cobamide remodeling enzymes or B12-binding domains and is instead a member of the apurinic/apyrimidinic (AP) endonuclease 2 enzyme superfamily. We speculate that CbiR enables bacteria to repurpose cobamides that they cannot otherwise use in order to grow under cobamide-requiring conditions; this function was confirmed by heterologous expression of cbiR in Escherichia coli Homologs of CbiR are found in over 200 microbial taxa across 22 phyla, suggesting that many bacteria may use CbiR to gain access to the diverse cobamides present in their environment.IMPORTANCE Cobamides, comprising the vitamin B12 family of cobalt-containing cofactors, are required for metabolism in all domains of life, including most bacteria. Cobamides have structural variability in the lower ligand, and selectivity for particular cobamides has been observed in most organisms studied to date. Here, we discovered that the beneficial human gut bacterium Akkermansia muciniphila can use a diverse range of cobamides due to its ability to change the cobamide structure via a process termed cobamide remodeling. We identify and characterize the novel enzyme CbiR that is necessary for initiating the cobamide remodeling process. The discovery of this enzyme has implications for understanding the ecological role of A. muciniphila in the gut and the functions of other bacteria that produce this enzyme.

Possible Precipitation of Acute Coronary Syndrome with Immune Checkpoint Blockade: A Case Report.

INTRODUCTION: Immune checkpoint inhibitors (ICI) have led to improved survival in patients with a number of different tumor types. The ICI agent nivolumab induces anti-tumor immune responses by inhibiting the programmed cell death 1 protein, but side effects include cardiac immune-related adverse events (irAE) such as myocarditis.¹ The association of nivolumab with atherosclerotic disease has been rarely reported. CASE PRESENTATION: A 62-year-old man with metastatic melanoma and recent myocardial infarction (MI) presented with recurrent MI after having undergone several cycles of nivolumab therapy. Repeat cardiac catheterization revealed rapidly progressive in-stent restenosis and diffuse coronary artery disease (CAD) requiring bypass surgery and warranting cessation of nivolumab therapy. CONCLUSION: Nivolumab has been linked with dysregulation of immune responses including enhanced T cell activity, which is implicated in CAD. The timing of nivolumab therapy and presentation with non ST elevation myocardial infarction in this patient suggests a serious T cell-driven medication adverse effect. Therefore, close monitoring for atherosclerotic disease progression is warranted in patients on immunotherapy.

Cofactor Selectivity in Methylmalonyl Coenzyme A Mutase, a Model Cobamide-Dependent Enzyme.

Cobamides, a uniquely diverse family of enzyme cofactors related to vitamin B12, are produced exclusively by bacteria and archaea but used in all domains of life. While it is widely accepted that cobamide-dependent organisms require specific cobamides for their metabolism, the biochemical mechanisms that make cobamides functionally distinct are largely unknown. Here, we examine the effects of cobamide structural variation on a model cobamide-dependent enzyme, methylmalonyl coenzyme A (CoA) mutase (MCM). The in vitro binding affinity of MCM for cobamides can be dramatically influenced by small changes in the structure of the lower ligand of the cobamide, and binding selectivity differs between bacterial orthologs of MCM. In contrast, variations in the lower ligand have minor effects on MCM catalysis. Bacterial growth assays demonstrate that cobamide requirements of MCM in vitro largely correlate with in vivo cobamide dependence. This result underscores the importance of enzyme selectivity in the cobamide-dependent physiology of bacteria.IMPORTANCE Cobamides, including vitamin B12, are enzyme cofactors used by organisms in all domains of life. Cobamides are structurally diverse, and microbial growth and metabolism vary based on cobamide structure. Understanding cobamide preference in microorganisms is important given that cobamides are widely used and appear to mediate microbial interactions in host-associated and aquatic environments. Until now, the biochemical basis for cobamide preferences was largely unknown. In this study, we analyzed the effects of the structural diversity of cobamides on a model cobamide-dependent enzyme, methylmalonyl-CoA mutase (MCM). We found that very small changes in cobamide structure could dramatically affect the binding affinity of cobamides to MCM. Strikingly, cobamide-dependent growth of a model bacterium, Sinorhizobium meliloti, largely correlated with the cofactor binding selectivity of S. meliloti MCM, emphasizing the importance of cobamide-dependent enzyme selectivity in bacterial growth and cobamide-mediated microbial interactions.

Uneven distribution of cobamide biosynthesis and dependence in bacteria predicted by comparative genomics.

The vitamin B12 family of cofactors known as cobamides are essential for a variety of microbial metabolisms. We used comparative genomics of 11,000 bacterial species to analyze the extent and distribution of cobamide production and use across bacteria. We find that 86% of bacteria in this data set have at least one of 15 cobamide-dependent enzyme families, but only 37% are predicted to synthesize cobamides de novo. The distribution of cobamide biosynthesis and use vary at the phylum level. While 57% of Actinobacteria are predicted to biosynthesize cobamides, only 0.6% of Bacteroidetes have the complete pathway, yet 96% of species in this phylum have cobamide-dependent enzymes. The form of cobamide produced by the bacteria could be predicted for 58% of cobamide-producing species, based on the presence of signature lower ligand biosynthesis and attachment genes. Our predictions also revealed that 17% of bacteria have partial biosynthetic pathways, yet have the potential to salvage cobamide precursors. Bacteria with a partial cobamide biosynthesis pathway include those in a newly defined, experimentally verified category of bacteria lacking the first step in the biosynthesis pathway. These predictions highlight the importance of cobamide and cobamide precursor salvaging as examples of nutritional dependencies in bacteria.

HM2-phage resistant solventogenic Clostridium saccharoperbutylacetonicum N1-4 shows increased exopolysaccharide production.

During the history of the solventogenic clostridia fermentation industry, bacteriophages have been a recurrent problem. This study reports that HM2, a lytic bacteriophage for solventogenic Clostridium saccharoperbutylacetonicum N1-4 (N1-4), has a genome size of 17 470 bp with 22 open reading frames, including replication, lysis, integration and structural modules. To understand HM2 infection and resistance in N1-4, bacteriophage insensitive mutants (BIMs) were isolated and characterized. These eight independent BIMs included four adsorption mediated and four non-adsorption types. Adsorption mediated BIMs had increased exopolysaccharide (EPS) production and decreased attachment of HM2 to the cell surface. Non-adsorption mediated BIMs had a moderate increase in EPS production, which did not affect HM2 attachment. Absorption mediated BIMs had reduced fermentation performance, whereas the non-absorption mediated BIMs were indistinguishable from N1-4. Consequently, non-adsorption mediated BIMs would be more useful in a fermentation since they couple good fermentation performance with phage resistance.

Sinorhizobium fredii†HH103 bacteroids are not terminally differentiated and show altered O-antigen in nodules of the Inverted Repeat-Lacking Clade legume Glycyrrhiza uralensis.

In rhizobial species that nodulate inverted repeat-lacking clade (IRLC) legumes, such as the interaction between Sinorhizobium meliloti and Medicago, bacteroid differentiation is driven by an endoreduplication event that is induced by host nodule-specific cysteine rich (NCR) antimicrobial peptides and requires the participation of the bacterial protein BacA. We have studied bacteroid differentiation of Sinorhizobium fredii HH103 in three host plants: Glycine max, Cajanus cajan and the IRLC legume Glycyrrhiza uralensis. Flow cytometry, microscopy analyses and viability studies of bacteroids as well as confocal microscopy studies carried out in nodules showed that S. fredii HH103 bacteroids, regardless of the host plant, had deoxyribonucleic acid (DNA) contents, cellular sizes and survival rates similar to those of free-living bacteria. Contrary to S. meliloti, S. fredii HH103 showed little or no sensitivity to Medicago NCR247 and NCR335 peptides. Inactivation of S. fredii HH103 bacA neither affected symbiosis with Glycyrrhiza nor increased bacterial sensitivity to Medicago NCRs. Finally, HH103 bacteroids isolated from Glycyrrhiza, but not those isolated from Cajanus or Glycine, showed an altered lipopolysaccharide. Our studies indicate that, in contrast to the S. meliloti-Medicago model symbiosis, bacteroids in the S. fredii HH103-Glycyrrhiza symbiosis do not undergo NCR-induced and bacA-dependent terminal differentiation.

Anaerobic biosynthesis of the lower ligand of vitamin B12.

Vitamin B12 (cobalamin) is required by humans and other organisms for diverse metabolic processes, although only a subset of prokaryotes is capable of synthesizing B12 and other cobamide cofactors. The complete aerobic and anaerobic pathways for the de novo biosynthesis of B12 are known, with the exception of the steps leading to the anaerobic biosynthesis of the lower ligand, 5,6-dimethylbenzimidazole (DMB). Here, we report the identification and characterization of the complete pathway for anaerobic DMB biosynthesis. This pathway, identified in the obligate anaerobic bacterium Eubacterium limosum, is composed of five previously uncharacterized genes, bzaABCDE, that together direct DMB production when expressed in anaerobically cultured Escherichia coli. Expression of different combinations of the bza genes revealed that 5-hydroxybenzimidazole, 5-methoxybenzimidazole, and 5-methoxy-6-methylbenzimidazole, all of which are lower ligands of cobamides produced by other organisms, are intermediates in the pathway. The bza gene content of several bacterial and archaeal genomes is consistent with experimentally determined structures of the benzimidazoles produced by these organisms, indicating that these genes can be used to predict cobamide structure. The identification of the bza genes thus represents the last remaining unknown component of the biosynthetic pathway for not only B12 itself, but also for three other cobamide lower ligands whose biosynthesis was previously unknown. Given the importance of cobamides in environmental, industrial, and human-associated microbial metabolism, the ability to predict cobamide structure may lead to an improved ability to understand and manipulate microbial metabolism.

Human gut microbes use multiple transporters to distinguish vitamin B12 analogs and compete in the gut.

Genomic and metagenomic sequencing efforts, including human microbiome projects, reveal that microbes often encode multiple systems that appear to accomplish the same task. Whether these predictions reflect actual functional redundancies is unclear. We report that the prominent human gut symbiont Bacteroides thetaiotaomicron employs three functional, homologous vitamin B12 transporters that in at least two cases confer a competitive advantage in the presence of distinct B12 analogs (corrinoids). In the mammalian gut, microbial fitness can be determined by the presence or absence of a single transporter. The total number of distinct corrinoid transporter families in the human gut microbiome likely exceeds those observed in B. thetaiotaomicron by an order of magnitude. These results demonstrate that human gut microbes use elaborate mechanisms to capture and differentiate corrinoids in vivo and that apparent redundancies observed in these genomes can instead reflect hidden specificities that determine whether a microbe will colonize its host.

Growth inhibition of Sporomusa ovata by incorporation of benzimidazole bases into cobamides.

Phenolyl cobamides are unique members of a class of cobalt-containing cofactors that includes vitamin B12 (cobalamin). Cobamide cofactors facilitate diverse reactions in prokaryotes and eukaryotes. Phenolyl cobamides are structurally and chemically distinct from the more commonly used benzimidazolyl cobamides such as cobalamin, as the lower axial ligand is a phenolic group rather than a benzimidazole. The functional significance of this difference is not well understood. Here we show that in the bacterium Sporomusa ovata, the only organism known to synthesize phenolyl cobamides, several cobamide-dependent acetogenic metabolisms have a requirement or preference for phenolyl cobamides. The addition of benzimidazoles to S. ovata cultures results in a decrease in growth rate when grown on methanol, 3,4-dimethoxybenzoate, H2 plus CO2, or betaine. Suppression of native p-cresolyl cobamide synthesis and production of benzimidazolyl cobamides occur upon the addition of benzimidazoles, indicating that benzimidazolyl cobamides are not functionally equivalent to the phenolyl cobamide cofactors produced by S. ovata. We further show that S. ovata is capable of incorporating other phenolic compounds into cobamides that function in methanol metabolism. These results demonstrate that S. ovata can incorporate a wide range of compounds as cobamide lower ligands, despite its preference for phenolyl cobamides in the metabolism of certain energy substrates. To our knowledge, S. ovata is unique among cobamide-dependent organisms in its preferential utilization of phenolyl cobamides.

Active site residues critical for flavin binding and 5,6-dimethylbenzimidazole biosynthesis in the flavin destructase enzyme BluB.

The "flavin destructase" enzyme BluB catalyzes the unprecedented conversion of flavin mononucleotide (FMN) to 5,6-dimethylbenzimidazole (DMB), a component of vitamin B(12). Because of its unusual chemistry, the mechanism of this transformation has remained elusive. This study reports the identification of 12 mutant forms of BluB that have severely reduced catalytic function, though most retain the ability to bind flavin. The "flavin destructase" BluB is an unusual enzyme that fragments the flavin cofactor FMNH(2) in the presence of oxygen to produce 5,6-dimethylbenzimidazole (DMB), the lower axial ligand of vitamin B(12) (cobalamin). Despite the similarities in sequence and structure between BluB and the nitroreductase and flavin oxidoreductase enzyme families, BluB is the only enzyme known to fragment a flavin isoalloxazine ring. To explore the catalytic residues involved in this unusual reaction, mutants of BluB impaired in DMB biosynthesis were identified in a genetic screen in the bacterium Sinorhizobium meliloti. Of the 16 unique point mutations identified in the screen, the majority were located in conserved residues in the active site or in the unique "lid" domain proposed to shield the active site from solvent. Steady-state enzyme assays of 12 purified mutant proteins showed a significant reduction in DMB synthesis in all of the mutants, with eight completely defective in DMB production. Ten of these mutants have weaker binding affinities for both oxidized and reduced FMN, though only two have a significant effect on complex stability. These results implicate several conserved residues in BluB's unique ability to fragment FMNH(2) and demonstrate the sensitivity of BluB's active site to structural perturbations. This work lays the foundation for mechanistic studies of this enzyme and further advances our understanding of the structure-function relationship of BluB.

The small RNA chaperone Hfq and multiple small RNAs control quorum sensing in Vibrio harveyi and Vibrio cholerae.

Quorum-sensing bacteria communicate with extracellular signal molecules called autoinducers. This process allows community-wide synchronization of gene expression. A screen for additional components of the Vibrio harveyi and Vibrio cholerae quorum-sensing circuits revealed the protein Hfq. Hfq mediates interactions between small, regulatory RNAs (sRNAs) and specific messenger RNA (mRNA) targets. These interactions typically alter the stability of the target transcripts. We show that Hfq mediates the destabilization of the mRNA encoding the quorum-sensing master regulators LuxR (V. harveyi) and HapR (V. cholerae), implicating an sRNA in the circuit. Using a bioinformatics approach to identify putative sRNAs, we identified four candidate sRNAs in V. cholerae. The simultaneous deletion of all four sRNAs is required to stabilize hapR mRNA. We propose that Hfq, together with these sRNAs, creates an ultrasensitive regulatory switch that controls the critical transition into the high cell density, quorum-sensing mode.

Vibrio harveyi quorum sensing: a coincidence detector for two autoinducers controls gene expression.

In a process called quorum sensing, bacteria communicate with one another by exchanging chemical signals called autoinducers. In the bioluminescent marine bacterium Vibrio harveyi, two different auto inducers (AI-1 and AI-2) regulate light emission. Detection of and response to the V.harveyi autoinducers are accomplished through two two-component sensory relay systems: AI-1 is detected by the sensor LuxN and AI-2 by LuxPQ. Here we further define the V.harveyi quorum-sensing regulon by identifying 10 new quorum-sensing-controlled target genes. Our examination of signal processing and integration in the V.harveyi quorum-sensing circuit suggests that AI-1 and AI-2 act synergistically, and that the V.harveyi quorum-sensing circuit may function exclusively as a 'coincidence detector' that discriminates between conditions in which both autoinducers are present and all other conditions.