Intratracheal cobinamide (vitamin B12 analog) administration increases survivability in rabbits exposed to a lethal dose of inhaled hydrogen sulfide.

BACKGROUND: Hydrogen sulfide is a highly toxic, flammable, and colorless gas. Hydrogen sulfide has been identified as a potential terrorist chemical threat agent in mass-casualty events. Our previous studies showed that cobinamide, a vitamin B12 analog, effectively reverses the toxicity from hydrogen sulfide poisoning. In this study, we investigate the effectiveness of intratracheally administered cobinamide in treating a lethal dose hydrogen sulfide gas inhalation and compare its performance to saline control administration. METHODS: A total of 53 pathogen-free New Zealand White rabbits were used for this study. Four groups were compared: (i) received no saline solution or drug intratracheally (n = 15), (ii) slow drip saline intratracheally (n = 15), (iii) fast drip saline intratracheally (n = 15), and (iv) slow drip cobinamide intratracheally (n = 8). Blood pressure was continuously monitored, and deoxy- and oxyhemoglobin concentration changes were monitored in real-time in vivo using continuous wave near-infrared spectroscopy. RESULTS: The mean (± standard deviation) weight for all animals (n = 53) was 3.87 ± 0.10 kg. The survival rates of the slow cobinamide and the fast saline groups were 75 percent and 60 percent, respectively, while the survival rates in the slow saline and control groups were 26.7 percent and 20 percent, respectively. A log-rank (Mantel-Cox) test showed that survival in fast saline and slow cobinamide groups were significantly greater than those of no saline control and slow saline groups (P < 0.05). The slow and no saline control groups were not significantly different (P = 0.59). The slow cobinamide group did significantly better than the slow saline group (P = 0.021). DISCUSSION: The ability to use intratracheal cobinamide as an antidote to hydrogen sulfide poisoning is a novel approach to mass-casualty care. The major limitations of this study are that it was conducted in a single species at a single inhaled hydrogen sulfide concentration. Repeated investigations in other species and at varying levels of hydrogen sulfide exposure will be needed before any definitive recommendations can be made. CONCLUSIONS: We demonstrated that intratracheal cobinamide and fast saline drip improved survival for hydrogen sulfide gas inhalation in rabbit models. Although further study is required, our results suggest that intratracheal administration of cobinamide and fast saline may be useful in hydrogen sulfide mass-casualty events.

Characterization of a Structurally Distinct ATP-Dependent Reactivating Factor of Adenosylcobalamin-Dependent Lysine 5,6-Aminomutase.

Several anaerobic bacterial species, including the Gram-negative oral bacterium Fusobacterium nucleatum, ferment lysine to produce butyrate, acetate, and ammonia. The second step of the metabolic pathway─isomerization of ß-l-lysine to erythro-3,5-diaminohexanoate─is catalyzed by the adenosylcobalamin (AdoCbl) and pyridoxal 5'-phosphate (PLP)-dependent enzyme, lysine 5,6-aminomutase (5,6-LAM). Similar to other AdoCbl-dependent enzymes, 5,6-LAM undergoes mechanism-based inactivation due to loss of the AdoCbl 5'-deoxyadenosyl moiety and oxidation of the cob(II)alamin intermediate to hydroxocob(III)alamin. Herein, we identified kamB and kamC, two genes responsible for ATP-dependent reactivation of 5,6-LAM. KamB and KamC, which are encoded upstream of the genes corresponding to α and ß subunits of 5,6-LAM (kamD and kamE), co-purified following coexpression of the genes in Escherichia coli. KamBC exhibited a basal level of ATP-hydrolyzing activity that was increased 35% in a reaction mixture that facilitated 5,6-LAM turnover with ß-l-lysine or d,l-lysine. Ultraviolet-visible (UV-vis) spectroscopic studies performed under anaerobic conditions revealed that KamBC in the presence of ATP/Mg2+ increased the steady-state concentration of the cob(II)alamin intermediate in the presence of excess ß-l-lysine. Using a coupled UV-visible spectroscopic assay, we show that KamBC is able to reactivate 5,6-LAM through exchange of the damaged hydroxocob(III)alamin for AdoCbl. KamBC is also specific for 5,6-LAM as it had no effect on the rate of substrate-induced inactivation of the homologue, ornithine 4,5-aminomutase. Based on sequence homology, KamBC is structurally distinct from previously characterized B12 chaperones and reactivases, and correspondingly adds to the list of proteins that have evolved to maintain the cellular activity of B12 enzymes.

Global biogeography and ecological implications of cobamide-producing prokaryotes.

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.

Tuning of B12 photochemistry in the CarH photoreceptor to avoid radical photoproducts.

Time-resolved infrared spectroscopy reveals the flow of electron density through coenzyme B12 in the light-activated, bacterial transcriptional regulator, CarH. The protein stabilises a series of charge transfer states that result in a photoresponse that avoids reactive, and potentially damaging, radical photoproducts.

Reactivity Tracking of an Enzyme Progress Coordinate.

The reactivity of individual solvent-coupled protein configurations is used to track and resolve the progress coordinate for the core reaction sequence of substrate radical rearrangement and hydrogen atom transfer in the ethanolamine ammonia-lyase (EAL) enzyme from Salmonella enterica. The first-order decay of the substrate radical intermediate is the monitored reaction. Heterogeneous confinement from sucrose hydrates in the mesophase solvent surrounding the cryotrapped protein introduces distributed kinetics in the non-native decay of the substrate radical pair capture substate, which arise from an ensemble of configurational microstates. Reaction rates increase by >103-fold across the distribution to approach that for the native enabled substate for radical rearrangement, which reacts with monotonic kinetics. The native progress coordinate thus involves a collapse of the configuration space to generate optimized reactivity. Reactivity tracking reveals fundamental features of solvent-protein-reaction configurational coupling and leads to a model that refines the ensemble paradigm of enzyme catalysis for strongly adiabatic chemical steps.

Metagenomic Evidence for Cobamide Producers Driving Prokaryotic Co-occurrence Associations and Potential Function in Wastewater Treatment Plants.

Cobamides are required by most organisms but are only produced by specific prokaryotic taxa. These commonly shared cofactors play significant roles in shaping the microbial community and ecosystem function. Wastewater treatment plants (WWTPs) are the world's most common biotechnological systems; knowledge about sharing of cobamides among microorganisms is predicted to be important to decipher the complex microbial relationships in these systems. Herein, we explored prokaryotic potential cobamide producers in global WWTP systems based on metagenomic analyses. A set of 8253 metagenome-assembled genomes (MAGs) were recovered and 1276 (15.5%) of them were identified as cobamide producers, which could potentially be used for the practical biological manipulation of WWTP systems. Moreover, 8090 of the total recovered MAGs (98.0%) contained at least one enzyme family dependent on cobamides, indicating the sharing of cobamides among microbial members in WWTP systems. Importantly, our results showed that the relative abundance and number of cobamide producers improved the complexity of microbial co-occurrence networks and most nitrogen, sulfur, and phosphorus cycling gene abundances, indicating the significance of cobamides in microbial ecology and their potential function in WWTP systems. These findings enhance the knowledge of cobamide producers and their functions in WWTP systems, which has important implications for improving the efficiency of microbial wastewater treatment processes.

Structural insight into G-protein chaperone-mediated maturation of a bacterial adenosylcobalamin-dependent mutase.

G-protein metallochaperones are essential for the proper maturation of numerous metalloenzymes. The G-protein chaperone MMAA in humans (MeaB in bacteria) uses GTP hydrolysis to facilitate the delivery of adenosylcobalamin (AdoCbl) to AdoCbl-dependent methylmalonyl-CoA mutase, an essential metabolic enzyme. This G-protein chaperone also facilitates the removal of damaged cobalamin (Cbl) for repair. Although most chaperones are standalone proteins, isobutyryl-CoA mutase fused (IcmF) has a G-protein domain covalently attached to its target mutase. We previously showed that dimeric MeaB undergoes a 180° rotation to reach a state capable of GTP hydrolysis (an active G-protein state), in which so-called switch III residues of one protomer contact the G-nucleotide of the other protomer. However, it was unclear whether other G-protein chaperones also adopted this conformation. Here, we show that the G-protein domain in a fused system forms a similar active conformation, requiring IcmF oligomerization. IcmF oligomerizes both upon Cbl damage and in the presence of the nonhydrolyzable GTP analog, guanosine-5'-[(ß,γ)-methyleno]triphosphate, forming supramolecular complexes observable by mass photometry and EM. Cryo-EM structural analysis reveals that the second protomer of the G-protein intermolecular dimer props open the mutase active site using residues of switch III as a wedge, allowing for AdoCbl insertion or damaged Cbl removal. With the series of structural snapshots now available, we now describe here the molecular basis of G-protein-assisted AdoCbl-dependent mutase maturation, explaining how GTP binding prepares a mutase for cofactor delivery and how GTP hydrolysis allows the mutase to capture the cofactor.

An Adenosylcobalamin Specific Whole-Cell Biosensor.

Vitamin B12 (cobalamin) is essential for human health and its deficiency results in anemia and neurological damage. Vitamin B12 exists in different forms with various bioactivity but most sensors are unable to discriminate between them. Here, a whole-cell agglutination assay that is specific for adenosylcobalamin (AboB12), which is one of two bioactive forms, is reported. This biosensor consists of Escherichia coli that express the AdoB12 specific binding domain of CarH at their surface. In the presence of AdoB12, CarH forms tetramers, which leads to specific bacterial cell-cell adhesions and agglutination. These CarH tetramers disassemble upon green light illumination such that reversion of the bacterial aggregation can serve as internal quality control. The agglutination assay has a detection limit of 500 nм AdoB12, works in protein-poor biofluids such as urine, and has high specificity to AdoB12 over other forms of vitamin B12 as also demonstrated with commercially available supplements. This work is a proof of concept for a cheap and easy-to-readout AdoB12 sensor that can be implemented at the point-of-care to monitor high-dose vitamin B12 supplementation.

The vitamin B12 analog cobinamide ameliorates azide toxicity in cells, Drosophila melanogaster, and mice.

CONTEXT: The azide anion (N3-) is highly toxic. It exists most commonly as sodium azide, which is used widely and is readily available, raising the potential for occupational incidents and use as a weapon of mass destruction. Azide-poisoned patients present with vomiting, seizures, hypotension, metabolic acidosis, and coma; death can occur. No specific azide antidote exists, with treatment being solely supportive. Azide inhibits mitochondrial cytochrome c oxidase and is likely oxidized to nitric oxide in vivo. Cytochrome c oxidase inhibition depletes intracellular adenosine triphosphate and increases oxidative stress, while increased nitric oxide causes hypotension and exacerbates oxidative damage. Here, we tested whether the cobalamin (vitamin B12) analog cobinamide, a strong and versatile antioxidant that also neutralizes nitric oxide, can reverse azide toxicity in mammalian cells, Drosophila melanogaster, and mice. RESULTS: We found cobinamide bound azide with a moderate affinity (Ka 2.87 × 105 M-1). Yet, cobinamide improved growth, increased intracellular adenosine triphosphate, and reduced apoptosis and malondialdehyde, a marker of oxidative stress, in azide-exposed cells. Cobinamide rescued Drosophila melanogaster and mice from lethal exposure to azide and was more effective than hydroxocobalamin. Azide likely generated nitric oxide in the mice, as evidenced by increased serum nitrite and nitrate, and reduced blood pressure and peripheral body temperature in the animals; the reduced temperature was likely due to reflex vasoconstriction in response to the hypotension. Cobinamide improved recovery of both blood pressure and body temperature. CONCLUSION: We conclude cobinamide likely acted by neutralizing both oxidative stress and nitric oxide, and that it should be given further consideration as an azide antidote.

Structural Basis for the Activation of the Cobalt-Carbon Bond and Control of the Adenosyl Radical in Coenzyme B12 Catalysis.

Adenosylcobalamin (AdoCbl), or coenzyme B12 , is a naturally occurring organometallic compound that serves as a cofactor for enzymes that catalyze intramolecular group-transfer reactions and ribonucleotide reduction in a wide variety of organisms from bacteria to animals. AdoCbl-dependent enzymes are radical enzymes that generate an adenosyl radical by homolysis of the coenzyme's cobalt-carbon (Co-C) bond for catalysis. How do the enzymes activate and cleave the Co-C bond to form the adenosyl radical? How do the enzymes utilize the high reactivity of the adenosyl radical for catalysis by suppressing undesirable side reactions? Our recent structural studies, which aimed to solve these problems with diol dehydratase and ethanolamine ammonia-lyase, established the crucial importance of the steric strain of the Co-C bond and conformational stabilization of the adenosyl radical for coenzyme B12 catalysis. We outline here our results obtained with these eliminating isomerases and compare them with those obtained with other radical B12 enzymes.

Nutrient encryption and the diversity of cobamides, siderophores, and glycans.

Encryption makes information available only to those with the decoding key. We propose that microbes, living in a chemical environment, encrypt nutrients, thereby making them available only to those with the decoding enzymes, such as their kin. Examples of encrypted nutrients include cobamides, which are expensive to make and valuable for microbial fitness. Furthermore, we propose that hosts encrypt nutrients to encourage desirable colonizers. For instance, plant root exudates and breast milk oligosaccharides encourage beneficial microbes.

Methylations in vitamin B12 biosynthesis and catalysis.

Vitamin B12 is an essential biomolecule that assists in the catalysis of methyl transfer and radical-based reactions in cellular metabolism. The structure of B12 is characterized by a tetrapyrrolic corrin ring with a central cobalt ion coordinated with an upper ligand, and a lower ligand anchored via a nucleotide loop. Multiple methyl groups decorate B12, and their presence (or absence) have structural and functional consequences. In this minireview, we focus on the methyl groups that distinguish vitamin B12 from other tetrapyrrolic biomolecules and from its own naturally occurring analogues called cobamides. We draw information from recent advances in the field to understand the origins of these methyl groups and the enzymes that incorporate them, and discuss their biological significance.

Structural studies of the phosphoribosyltransferase involved in cobamide biosynthesis in methanogenic archaea and cyanobacteria.

Cobamides (Cbas) are coenzymes used by cells across all domains of life, but de novo synthesis is only found in some bacteria and archaea. Five enzymes assemble the nucleotide loop in the alpha phase of the corrin ring. Condensation of the activated ring and nucleobase yields adenosyl-Cba 5'-phosphate, which upon dephosphorylation yields the biologically active coenzyme (AdoCba). Base activation is catalyzed by a phosphoribosyltransferase (PRTase). The structure of the Salmonella enterica PRTase enzyme (i.e., SeCobT) is well-characterized, but archaeal PRTases are not. To gain insights into the mechanism of base activation by the PRTase from Methanocaldococcus jannaschii (MjCobT), we solved crystal structures of the enzyme in complex with substrate and products. We determined several structures: (i) a 2.2 Å structure of MjCobT in the absence of ligand (apo), (ii) structures of MjCobT bound to nicotinate mononucleotide (NaMN) and α-ribazole 5'-phosphate (α-RP) or α-adenylyl-5'-phosphate (α-AMP) at 2.3 and 1.4 Å, respectively. In MjCobT the general base that triggers the reaction is an aspartate residue (Asp 52) rather than a glutamate residue (E317) as in SeCobT. Notably, the dimer interface in MjCobT is completely different from that observed in SeCobT. Finally, entry PDB 3L0Z does not reflect the correct structure of MjCobT.

Structural Insights into the Very Low Activity of the Homocoenzyme B12 Adenosylmethylcobalamin in Coenzyme B12 -Dependent Diol Dehydratase and Ethanolamine Ammonia-Lyase.

The X-ray structures of coenzyme B12 (AdoCbl)-dependent eliminating isomerases complexed with adenosylmethylcobalamin (AdoMeCbl) have been determined. As judged from geometries, the Co-C bond in diol dehydratase (DD) is not activated even in the presence of substrate. In ethanolamine ammonia-lyase (EAL), the bond is elongated in the absence of substrate; in the presence of substrate, the complex likely exists in both pre- and post-homolysis states. The impacts of incorporating an extra CH2 group are different in the two enzymes: the DD active site is flexible, and AdoMeCbl binding causes large conformational changes that make DD unable to adopt the catalytic state, whereas the EAL active site is rigid, and AdoMeCbl binding does not induce significant conformational changes. Such flexibility and rigidity of the active sites might reflect the tightness of adenine binding. The structures provide good insights into the basis of the very low activity of AdoMeCbl in these enzymes.

A Salvaging Strategy Enables Stable Metabolite Provisioning among Free-Living Bacteria.

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.

Analysis of bisaminotetrazole cobinamide, a next-generation antidote for cyanide, hydrogen sulfide and methanethiol poisoning, in swine plasma by liquid chromatography-tandem mass spectrometry.

Cyanide, hydrogen sulfide, and methanethiol are common toxic inhalation agents that inhibit mitochondrial cytochrome c oxidase and result in cellular hypoxia, cytotoxic anoxia, apnea, respiratory failure, cardiovascular collapse, seizure and potentially death. While all are occupational gas exposure hazards that have the potential to cause mass casualties from industrial accidents or acts of terrorism, only cyanide has approved antidotes, and each of these has major limitations, including difficult administration in mass-casualty settings. While bisaminotetrazole cobinamide (Cbi(AT)2) has recently gained attention because of its efficacy in treating these metabolic poisons, there is no method available for the analysis of Cbi(AT)2 in any biological matrix. Hence, in this study, a simple and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the analysis of Cbi(AT)2 in swine plasma. The method is extremely simple, consisting of protein precipitation, separation and drying of the supernatant, reconstitution in an aqueous solvent, and LC-MS/MS analysis. The method produced an LOD of 0.3 µM with a wide dynamic range (2 - 500 µM). Inter- and intraassay accuracies (100 ± 12 % and 100 ± 19 %, respectively) were acceptable and the precision (<12 % and < 9 % relative standard deviation, respectively) was good. The developed method was used to analyze Cbi(AT)2 from treated swine and the preliminary pharmacokinetic parameters showed impressive antidotal behavior, most notably a long estimated elimination half-life (t1/2 = 37.5 h). This simple and rapid method can be used to facilitate the development of Cbi(AT)2 as a therapeutic against toxic cyanide, hydrogen sulfide and methanethiol exposure.

Cobamide Sharing Is Predicted in the Human Skin Microbiome.

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.

Comparative Analysis of Corrinoid Profiles across Host-Associated and Environmental Samples.

Vitamin B12 (the cyanated form of cobalamin cofactors) is best known for its essential role in human health. In addition to its function in human metabolism, cobalamin also plays important roles in microbial metabolism and can impact microbial community function. Cobalamin is a member of the structurally diverse family of cofactors known as cobamides that are produced exclusively by certain prokaryotes. Cobamides are considered shared nutrients in microbial communities because the majority of bacteria that possess cobamide-dependent enzymes cannot synthesize cobamides de novo. Furthermore, different microbes have evolved metabolic specificity for particular cobamides, and therefore, the availability of cobamides in the environment is important for cobamide-dependent microbes. Determining the cobamides present in an environment of interest is essential for understanding microbial metabolic interactions. By examining the abundances of different cobamides in diverse environments, including 10 obtained in this study, we find that, contrary to its preeminence in human metabolism, cobalamin is relatively rare in many microbial habitats. Comparison of cobamide profiles of mammalian gastrointestinal samples and wood-feeding insects reveals that host-associated cobamide abundances vary and that fecal cobamide profiles differ from those of their host gastrointestinal tracts. Environmental cobamide profiles obtained from aquatic, soil, and contaminated groundwater samples reveal that the cobamide compositions of environmental samples are highly variable. As the only commercially available cobamide, cobalamin is routinely supplied during microbial culturing efforts. However, these findings suggest that cobamides specific to a given microbiome may yield greater insight into nutrient utilization and physiological processes that occur in these habitats.

Acinetobacter baumannii Catabolizes Ethanolamine in the Absence of a Metabolosome and Converts Cobinamide into Adenosylated Cobamides.

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.