Acinetobacter baumannii is an opportunistic pathogen typically associated with hospital-acquired infections. Our understanding of the metabolism and physiology of A. baumannii is limited. Here, we report that A. baumannii uses ethanolamine (EA) as the sole source of nitrogen and can use this aminoalcohol as a source of carbon and energy if the expression of the eutBC genes encoding ethanolamine ammonia-lyase (EAL) is increased. A strain with an ISAba1 element upstream of the eutBC genes efficiently used EA as a carbon and energy source. The A. baumannii EAL (AbEAL) enzyme supported the growth of a strain of Salmonella lacking the entire eut operon. Remarkably, the growth of the above-mentioned Salmonella strain did not require the metabolosome, the reactivase EutA enzyme, the EutE acetaldehyde dehydrogenase, or the addition of glutathione to the medium. Transmission electron micrographs showed that when Acinetobacter baumannii or Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 synthesized AbEAL, the protein localized to the cell membrane. We also report that the A. baumannii genome encodes all of the enzymes needed for the assembly of the nucleotide loop of cobamides and that it uses these enzymes to synthesize different cobamides from the precursor cobinamide and several nucleobases. In the absence of exogenous nucleobases, the most abundant cobamide produced by A. baumannii was cobalamin. IMPORTANCE Acinetobacter baumannii is a Gram-negative bacterium commonly found in soil and water. A. baumannii is an opportunistic human pathogen, considered by the CDC to be a serious threat to human health due to the multidrug resistance commonly associated with this bacterium. Knowledge of the metabolic capabilities of A. baumannii is limited. The importance of the work reported here lies in the identification of ethanolamine catabolism occurring in the absence of a metabolosome structure. In other bacteria, this structure protects the cell against damage by acetaldehyde generated by the deamination of ethanolamine. In addition, the ethanolamine ammonia-lyase (EAL) enzyme of this bacterium is unique in that it does not require a reactivase enzyme to remain active. Importantly, we also demonstrate that the A. baumannii genome encodes the functions needed to assemble adenosylcobamide, the coenzyme of EAL, from the precursor cobinamide.

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.

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.

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.

Cobamides are a family of enzyme cofactors that include vitamin B12 (cobalamin) and are produced solely by prokaryotes. Structural variability in the lower axial ligand has been observed in cobamides produced by diverse organisms. Of the three classes of lower ligands, the benzimidazoles are uniquely found in cobamides, whereas the purine and phenolic bases have additional biological functions. Many organisms acquire cobamides by salvaging and remodeling cobamides or their precursors from the environment. These processes require free benzimidazoles for incorporation as lower ligands, though the presence of benzimidazoles in the environment has not been previously investigated. Here, we report a new purification method and bioassay to measure the total free benzimidazole content of samples from microbial communities and laboratory media components. The bioassay relies on the "calcofluor-bright" phenotype of a bluB mutant of the model cobalamin-producing bacterium Sinorhizobium meliloti. The concentrations of individual benzimidazoles in these samples were measured by liquid chromatography-tandem mass spectrometry. Several benzimidazoles were detected in subpicomolar to subnanomolar concentrations in host-associated and environmental samples. In addition, benzimidazoles were found to be common contaminants of laboratory media components. These results suggest that benzimidazoles present in the environment and in laboratory media have the potential to influence microbial metabolic activities.

Cobamides such as vitamin B12 are structurally conserved, cobalt-containing tetrapyrrole biomolecules that have essential biochemical functions in all domains of life. In organohalide respiration, a vital biological process for the global cycling of natural and anthropogenic organohalogens, cobamides are the requisite prosthetic groups for carbon-halogen bond-cleaving reductive dehalogenases. This study reports the biosynthesis of a new cobamide with unsubstituted purine as the lower base and assigns unsubstituted purine a biological function by demonstrating that Coα-purinyl-cobamide (purinyl-Cba) is the native prosthetic group in catalytically active tetrachloroethene reductive dehalogenases of Desulfitobacterium hafniense. Cobamides featuring different lower bases are not functionally equivalent, and purinyl-Cba elicits different physiological responses in corrinoid-auxotrophic, organohalide-respiring bacteria. Given that cobamide-dependent enzymes catalyze key steps in essential metabolic pathways, the discovery of a novel cobamide structure and the realization that lower bases can effectively modulate enzyme activities generate opportunities to manipulate functionalities of microbiomes.

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.

Cobamide diversity arises from the nature of the nucleotide base. Nicotinate mononucleotide (NaMN):base phosphoribosyltransferases (CobT) synthesize α-linked riboside monophosphates from diverse nucleotide base substrates (e.g., benzimidazoles, purines, phenolics) that are incorporated into cobamides.

Enzymes catalyze a wide variety of reactions with exquisite precision under crowded conditions within cellular environments. When encountered with a choice of small molecules in their vicinity, even though most enzymes continue to be specific about the substrate they pick, some others are able to accept a range of substrates and subsequently produce a variety of products. The biosynthesis of Vitamin B12, an essential nutrient required by humans involves a multi-substrate α-phosphoribosyltransferase enzyme CobT that activates the lower ligand of B12. Vitamin B12 is a member of the cobamide family of cofactors which share a common tetrapyrrolic corrin scaffold with a centrally coordinated cobalt ion, and an upper and a lower ligand. The structural difference between B12 and other cobamides mainly arises from variations in the lower ligand, which is attached to the activated corrin ring by CobT and other downstream enzymes. In this chapter, we describe the steps involved in identifying and reconstituting the activity of new CobT homologs by deriving lessons from those previously characterized. We then highlight biochemical techniques to study the unique properties of these homologs. Finally, we describe a pairwise substrate competition assay to rank CobT substrate preference, a general method that can be applied for the study of other multi-substrate enzymes. Overall, the analysis with CobT provides insights into the range of cobamides that can be synthesized by an organism or a community, complementing efforts to predict cobamide diversity from complex metagenomic data.

Vitamin B12 and other cobamides are essential cofactors required by many organisms and are synthesized by a subset of prokaryotes via distinct aerobic and anaerobic routes. The anaerobic biosynthesis of 5,6-dimethylbenzimidazole (DMB), the lower ligand of vitamin B12, involves five reactions catalyzed by the bza operon gene products, namely the hydroxybenzimidazole synthase BzaAB/BzaF, phosphoribosyltransferase CobT, and three methyltransferases, BzaC, BzaD, and BzaE, that conduct three distinct methylation steps. Of these, the methyltransferases that contribute to benzimidazole lower ligand diversity in cobamides remain to be characterized, and the precise role of the bza operon protein CobT is unclear. In this study, we used the bza operon from the anaerobic bacterium Moorella thermoacetica (comprising bzaA-bzaB-cobT-bzaC) to examine the role of CobT and investigate the activity of the first methyltransferase, BzaC. We studied the phosphoribosylation catalyzed by MtCobT and found that it regiospecifically activates 5-hydroxybenzimidazole (5-OHBza) to form the 5-OHBza-ribotide (5-OHBza-RP) isomer as the sole product. Next, we characterized the domains of MtBzaC and reconstituted its methyltransferase activity with the predicted substrate 5-OHBza and with two alternative substrates, the MtCobT product 5-OHBza-RP and its riboside derivative 5-OHBza-R. Unexpectedly, we found that 5-OHBza-R is the most favored MtBzaC substrate. Our results collectively explain the long-standing observation that the attachment of the lower ligand in anaerobic cobamide biosynthesis is regiospecific. In conclusion, we validate MtBzaC as a SAM:hydroxybenzimidazole-riboside methyltransferase (HBIR-OMT). Finally, we propose a new pathway for the synthesis and activation of the benzimidazolyl lower ligand in anaerobic cobamide biosynthesis.

The skin microbiome is a key player in human health, with diverse functions ranging from defense against pathogens to education of the immune system. While recent studies have begun to shed light on the valuable role that skin microorganisms have in maintaining the skin barrier, a detailed understanding of the complex interactions that shape healthy skin microbial communities is limited. Cobamides, the vitamin B12 class of cofactor, are essential for organisms across the tree of life. Because this vitamin is only produced by a limited fraction of prokaryotes, cobamide sharing is predicted to mediate community dynamics within microbial communities. Here, we provide the first large-scale metagenomic assessment of cobamide biosynthesis and utilization in the skin microbiome. We show that while numerous and diverse taxa across the major bacterial phyla on the skin encode cobamide-dependent enzymes, relatively few species encode de novo cobamide biosynthesis. We show that cobamide producers and users are integrated into the network structure of microbial communities across the different microenvironments of the skin and that changes in microbiome community structure and diversity are associated with the abundance of cobamide producers in the Corynebacterium genus, for both healthy and diseased skin states. Finally, we find that de novo cobamide biosynthesis is enriched only in Corynebacterium species associated with hosts, including those prevalent on human skin. We confirm that the cofactor is produced in excess through quantification of cobamide production by human skin-associated species isolated in the laboratory. Taken together, our results reveal the potential for cobamide sharing within skin microbial communities, which we hypothesize mediates microbiome community dynamics and host interactions. IMPORTANCE The skin microbiome is essential for maintaining skin health and function. However, the microbial interactions that dictate microbiome structure, stability, and function are not well understood. Here, we investigate the biosynthesis and use of cobamides, a cofactor needed by many organisms but only produced by select prokaryotes, within the human skin microbiome. We found that while a large proportion of skin taxa encode cobamide-dependent enzymes, only a select few encode de novo cobamide biosynthesis. Further, the abundance of cobamide-producing Corynebacterium species is associated with skin microbiome diversity and structure, and within this genus, de novo biosynthesis is enriched in host-associated species compared to environment-associated species. These findings identify cobamides as a potential mediator of skin microbiome dynamics and skin health.

Cobamides, a class of essential coenzymes synthesized only by a subset of prokaryotes, are model nutrients in microbial interaction studies and play significant roles in global ecosystems. Yet, their spatial patterns and functional roles remain poorly understood. Herein, we present an in-depth examination of cobamide-producing microorganisms, drawn from a comprehensive analysis of 2862 marine and 2979 soil metagenomic samples. A total of 1934 nonredundant metagenome-assembled genomes (MAGs) potentially capable of producing cobamides de novo were identified. The cobamide-producing MAGs are taxonomically diverse but habitat specific. They constituted only a fraction of all the recovered MAGs, with the majority of MAGs being potential cobamide users. By mapping the distribution of cobamide producers in marine and soil environments, distinct latitudinal gradients were observed: the marine environment showed peak abundance at the equator, whereas soil environments peaked at mid-latitudes. Importantly, significant and positive links between the abundance of cobamide producers and the diversity and functions of microbial communities were observed, as well as their promotional roles in essential biogeochemical cycles. These associations were more pronounced in marine samples than in soil samples, which suggests a heightened propensity for microorganisms to engage in cobamide sharing in fluid environments relative to the more spatially restricted soil environment. These findings shed light on the global patterns and potential ecological roles of cobamide-producing microorganisms in marine and soil ecosystems, enhancing our understanding of large-scale microbial interactions.

Cobamides are cobalt-containing cyclic tetrapyrroles involved in the metabolism of organisms from all domains of life but produced de novo only by some bacteria and archaea. The pathway is thought to involve up to 30 enzymes, five of which comprise the so-called "late" steps of cobamide biosynthesis. Two of these reactions activate the corrin ring, one activates the nucleobase, a fourth one condenses activated precursors, and a phosphatase yields the final product of the pathway. The penultimate step is catalyzed by a polytopic integral membrane protein, namely, the cobamide (5'-phosphate) synthase, also known as cobamide synthase. At present, the reason for the association of all putative and bona fide cobamide synthases to cell membranes is unclear and intriguing. Here, we show that, in Escherichia coli, elevated levels of cobamide synthase kill the cell by dissipating the proton motive force and compromising membrane stability. We also show that overproduction of the phosphatase that catalyzes the last step of the pathway or phage shock protein A prevents cell death when the gene encoding cobamide synthase is overexpressed. We propose that in E. coli, and probably all cobamide producers, cobamide synthase anchors a multienzyme complex responsible for the assembly of vitamin B12 and other cobamides. IMPORTANCE E. coli is the best-studied prokaryote, and some strains of this bacterium are human pathogens. We show that when the level of the enzyme that catalyzes the penultimate step of vitamin B12 biosynthesis is elevated, the viability of E. coli decreases. These findings are of broad significance because the enzyme alluded to is an integral membrane protein in all cobamide-producing bacteria, many of which are human pathogens. Our results may provide new avenues for the development of antimicrobials, because none of the enzymes involved in vitamin B12 biosynthesis are present in mammalian cells.

MetaCyc (MetaCyc.org) is a comprehensive reference database of metabolic pathways and enzymes from all domains of life. It contains 2749 pathways derived from more than 60 000 publications, making it the largest curated collection of metabolic pathways. The data in MetaCyc are evidence-based and richly curated, resulting in an encyclopedic reference tool for metabolism. MetaCyc is also used as a knowledge base for generating thousands of organism-specific Pathway/Genome Databases (PGDBs), which are available in BioCyc.org and other genomic portals. This article provides an update on the developments in MetaCyc during September 2017 to August 2019, up to version 23.1. Some of the topics that received intensive curation during this period include cobamides biosynthesis, sterol metabolism, fatty acid biosynthesis, lipid metabolism, carotenoid metabolism, protein glycosylation, antibiotics and cytotoxins biosynthesis, siderophore biosynthesis, bioluminescence, vitamin K metabolism, brominated compound metabolism, plant secondary metabolism and human metabolism. Other additions include modifications to the GlycanBuilder software that enable displaying glycans using symbolic representation, improved graphics and fonts for web displays, improvements in the PathoLogic component of Pathway Tools, and the optional addition of regulatory information to pathway diagrams.

Cobamides (Cbas) are essential cofactors of reductive dehalogenases (RDases) in organohalide-respiring bacteria (OHRB). Changes in the Cba structure can influence RDase function. Here, we report on the cofactor versatility or selectivity of Desulfitobacterium RDases produced either in the native organism or heterologously. The susceptibility of Desulfitobacterium hafniense strain DCB-2 to guided Cba biosynthesis (i.e. incorporation of exogenous Cba lower ligand base precursors) was analysed. Exogenous benzimidazoles, azabenzimidazoles and 4,5-dimethylimidazole were incorporated by the organism into Cbas. When the type of Cba changed, no effect on the turnover rate of the 3-chloro-4-hydroxy-phenylacetate-converting enzyme RdhA6 and the 3,5-dichlorophenol-dehalogenating enzyme RdhA3 was observed. The impact of the amendment of Cba lower ligand precursors on RDase function was also investigated in Shimwellia blattae, the Cba producer used for the heterologous production of Desulfitobacterium RDases. The recombinant tetrachloroethene RDase (PceAY51 ) appeared to be non-selective towards different Cbas. However, the functional production of the 1,2-dichloroethane-dihaloeliminating enzyme (DcaA) of Desulfitobacterium dichloroeliminans was completely prevented in cells producing 5,6-dimethylbenzimidazolyl-Cba, but substantially enhanced in cells that incorporated 5-methoxybenzimidazole into the Cba cofactor. The results of the study indicate the utilization of a range of different Cbas by Desulfitobacterium RDases with selected representatives apparently preferring distinct Cbas.

Bacteria in the genus Geobacter thrive in iron- and manganese-rich environments where the divalent cobalt cation (CoII) accumulates to potentially toxic concentrations. Consistent with selective pressure from environmental exposure, the model laboratory representative Geobacter sulfurreducens grew with CoCl2 concentrations (1 mM) typically used to enrich for metal-resistant bacteria from contaminated sites. We reconstructed from genomic data canonical pathways for CoII import and assimilation into cofactors (cobamides) that support the growth of numerous syntrophic partners. We also identified several metal efflux pumps, including one that was specifically upregulated by CoII. Cells acclimated to metal stress by downregulating non-essential proteins with metals and thiol groups that CoII preferentially targets. They also activated sensory and regulatory proteins involved in detoxification as well as pathways for protein and DNA repair. In addition, G. sulfurreducens upregulated respiratory chains that could have contributed to the reductive mineralization of the metal on the cell surface. Transcriptomic evidence also revealed pathways for cell envelope modification that increased metal resistance and promoted cell-cell aggregation and biofilm formation in stationary phase. These complex adaptive responses confer on Geobacter a competitive advantage for growth in metal-rich environments that are essential to the sustainability of cobamide-dependent microbiomes and the sequestration of the metal in hitherto unknown biomineralization reactions.

Human gut commensal Bacteroidetes rely on multiple transport systems to acquire vitamin B12 and related cobamides for fitness in the gut. In addition to a set of conserved transport proteins, these systems also include a diverse repertoire of additional proteins with unknown function. Here, we report the function and structural characterization of one of these proteins, BtuH, which binds vitamin B12 directly via a C-terminal globular domain that has no known structural homologs. This protein is required for efficient B12 transport and competitive fitness in the gut, demonstrating that members of the heterogeneous suite of accessory proteins encoded in Bacteroides cobamide transport system loci can play key roles in vitamin acquisition. IMPORTANCE The gut microbiome is a complex microbial community with important impacts on human health. One of the major groups within the gut microbiome, the Bacteroidetes, rely on their ability to capture vitamin B12 and related molecules for fitness in the gut. Unlike well-studied model organisms, gut Bacteroidetes genomes often include multiple vitamin B12 transport systems with a heterogeneous set of components. The role, if any, of these components was unknown. Here, we identify new proteins that play key roles in vitamin B12 capture in these organisms. Notably, these proteins are associated with some B12 transport systems and not others (even in the same bacterial strain), suggesting that these systems may assemble into functionally distinct machines to capture vitamin B12 and related molecules.

All organisms rely on complex metabolites such as amino acids, nucleotides, and cofactors for essential metabolic processes. Some microbes synthesize these fundamental ingredients of life de novo, while others rely on uptake to fulfill their metabolic needs. Although certain metabolic processes are inherently "leaky," the mechanisms enabling stable metabolite provisioning among microbes in the absence of a host remain largely unclear. In particular, how can metabolite provisioning among free-living bacteria be maintained under the evolutionary pressure to economize resources? Salvaging, the process of "recycling and reusing," can be a metabolically efficient route to obtain access to required resources. Here, we show experimentally how precursor salvaging in engineered Escherichia coli populations can lead to stable, long-term metabolite provisioning. We find that salvaged cobamides (vitamin B12 and related enzyme cofactors) are readily made available to nonproducing population members, yet salvagers are strongly protected from overexploitation. We also describe a previously unnoted benefit of precursor salvaging, namely, the removal of the nonfunctional, proliferation-inhibiting precursor. As long as compatible precursors are present, any microbe possessing the terminal steps of a biosynthetic process can, in principle, forgo de novo biosynthesis in favor of salvaging. Consequently, precursor salvaging likely represents a potent, yet overlooked, alternative to de novo biosynthesis for the acquisition and provisioning of metabolites in free-living bacterial populations. IMPORTANCE Recycling gives new life to old things. Bacteria have the ability to recycle and reuse complex molecules they encounter in their environment to fulfill their basic metabolic needs in a resource-efficient way. By studying the salvaging (recycling and reusing) of vitamin B12 precursors, we found that metabolite salvaging can benefit others and provide stability to a bacterial community at the same time. Salvagers of vitamin B12 precursors freely share the result of their labor yet cannot be outcompeted by freeloaders, likely because salvagers retain preferential access to the salvaging products. Thus, salvaging may represent an effective, yet overlooked, mechanism of acquiring and provisioning nutrients in microbial populations.

Cobamides are cobalt-containing cyclic tetrapyrroles used by cells from all domains of life but only produced de novo by some bacteria and archaea. The "late steps" of the adenosylcobamide biosynthetic pathway are responsible for the assembly of the nucleotide loop and are required during de novo synthesis and precursor salvaging. These steps are characterized by activation of the corrin ring and lower ligand base, condensation of the activated precursors to adenosylcobamide phosphate, and removal of the phosphate, yielding a complete adenosylcobamide molecule. The condensation of the activated corrin ring and lower ligand base is performed by an integral membrane protein, cobamide (5' phosphate) synthase (CobS), and represents an important convergence of two pathways necessary for nucleotide loop assembly. Interestingly, membrane association of this penultimate step is conserved among all cobamide producers, yet the physiological relevance of this association is not known. Here, we present the purification and biochemical characterization of the CobS enzyme of the enterobacterium Salmonella enterica subsp. enterica serovar Typhimurium strain LT2, investigate its association with liposomes, and quantify the effect of the lipid bilayer on its enzymatic activity and substrate affinity. We report a purification scheme that yields pure CobS protein, allowing in vitro functional analysis. Additionally, we report a method for liposome reconstitution of CobS, allowing for physiologically relevant studies of this inner membrane protein in a phospholipid bilayer. In vitro and in vivo data reported here expand our understanding of CobS and the implications of membrane-associated adenosylcobamide biosynthesis.IMPORTANCESalmonella is a human pathogen of worldwide importance, and coenzyme B12 is critical for the pathogenic lifestyle of this bacterium. The importance of the work reported here lies on the improvements to the methodology used to isolate cobamide synthase, a polytopic integral membrane protein that catalyzes the penultimate step of coenzyme B12 biosynthesis. This advance is an important step in the analysis of the proposed multienzyme complex responsible for the assembly of the nucleotide loop during de novo coenzyme B12 biosynthesis and for the assimilation of incomplete corrinoids from the environment. We proposed that cobamide synthase is likely localized to the cell membrane of every coenzyme B12-producing bacterium and archaeum sequenced to date. The new knowledge of cobamide synthase advances our understanding of the functionality of the enzyme in the context of the lipid bilayer and sets the foundation for the functional-structural analysis of the aforementioned multienzyme complex.