Aerobic adaptation and metabolic dynamics of Propionibacterium freudenreichii DSM 20271: insights from comparative transcriptomics and surfaceome analysis.

Propionibacterium freudenreichii (PFR) DSM 20271T is a bacterium known for its ability to thrive in diverse environments and to produce vitamin B12. Despite its anaerobic preference, recent studies have elucidated its ability to prosper in the presence of oxygen, prompting a deeper exploration of its physiology under aerobic conditions. Here, we investigated the response of DSM 20271T to aerobic growth by employing comparative transcriptomic and surfaceome analyses alongside metabolite profiling. Cultivation under controlled partial pressure of oxygen (pO2) conditions revealed significant increases in biomass formation and altered metabolite production, notably of vitamin B12, pseudovitamin-B12, propionate, and acetate, under aerobic conditions. Transcriptomic analysis identified differential expression of genes involved in lactate metabolism, tricarboxylic acid cycle, and electron transport chain, suggesting metabolic adjustments to aerobic environments. Moreover, surfaceome analysis unveiled growth environment-dependent changes in surface protein abundance, with implications for adaptation to atmospheric conditions. Supplementation experiments with key compounds highlighted the potential for enhancing aerobic growth, emphasizing the importance of iron and α-ketoglutarate availability. Furthermore, in liquid culture, FeSO4 supplementation led to increased heme production and reduced vitamin B12 production, highlighting the impact of oxygen and iron availability on the metabolic pathways. These findings deepen our understanding of PFR's physiological responses to oxygen availability and offer insights for optimizing its growth in industrial applications. IMPORTANCE: The study of the response of Propionibacterium freudenreichii to aerobic growth is crucial for understanding how this bacterium adapts to different environments and produces essential compounds like vitamin B12. By investigating its physiological changes under aerobic conditions, we can gain insights into its metabolic adjustments and potential for enhanced growth. These findings not only deepen our understanding of P. freudenreichii's responses to oxygen availability but also offer valuable information for optimizing its growth in industrial applications. This research sheds light on the adaptive mechanisms of this bacterium, providing a foundation for further exploration and potential applications in various fields.

Understanding the sources, function, and irreplaceable role of cobamides in organohalide-respiring bacteria.

Halogenated organic compounds are persistent pollutants that pose a serious threat to human health and the safety of ecosystems. Cobamides are essential cofactors for reductive dehalogenases (RDase) in organohalide-respiring bacteria (OHRB), which catalyze the dehalogenation process. This review systematically summarizes the impact of cobamides on organohalide respiration. The catalytic processes of cobamide in dehalogenation processes are also discussed. Additionally, we examine OHRB, which cannot synthesize cobamide and must obtain it from the environment through a salvage pathway; the co-culture with cobamide producer is more beneficial and possible. This review aims to help readers better understand the importance and function of cobamides in reductive dehalogenation. The presented information can aid in the development of bioremediation strategies.

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.

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.

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.

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.

Effects of cyanocobalamin and its combination with morphine on neuropathic rats and the relationship between these effects and thrombospondin-4 expression.

BACKGROUND: Thrombospondin-4 (TSP4) upregulates in the spinal cord following peripheral nerve injury and contributes to the development of neuropathic pain (NP). We investigated the effects of cyanocobalamin alone or in combination with morphine on pain and the relationship between these effects and spinal TSP4 expression in neuropathic rats. METHODS: NP was induced by chronic constriction injury (CCI) of the sciatic nerve. Cyanocobalamin (5 and 10 mg/kg/day) was administered 15 days before CCI and then for 4 and 14 postoperative days. Morphine (2.5 and 5 mg/kg/day) was administered only post-CCI. Combination treatment included cyanocobalamin and morphine, 10 and 5 mg/kg/day, respectively. All drugs were administered intraperitoneally. Nociceptive thresholds were detected by esthesiometer, analgesia meter, and plantar test, and TSP4 expression was assessed by western blotting and fluorescence immunohistochemistry. RESULTS: CCI decreased nociceptive thresholds in all tests and induced TSP4 expression on the 4th postoperative day. The decrease in nociceptive thresholds persisted except for the plantar test, and the increased TSP4 expression reversed on the 14th postoperative day. Cyanocobalamin and low-dose morphine alone did not produce any antinociceptive effects. High-dose morphine improved the decreased nociceptive thresholds in the esthesiometer when administered alone but combined with cyanocobalamin in all tests. Cyanocobalamin and morphine significantly induced TSP4 expression when administered alone in both doses for 4 or 14 days. However, this increase was less when the two drugs are combined. CONCLUSIONS: The combination of cyanocobalamin and morphine is more effective in antinociception and partially decreased the induced TSP4 expression compared to the use of either drug alone.

Nutrient smuggling: Commensal gut bacteria-derived extracellular vesicles scavenge vitamin B12 and related cobamides for microbe and host acquisition.

The processes by which bacteria proactively scavenge essential nutrients in crowded environments such as the gastrointestinal tract are not fully understood. In this context, we observed that bacterial extracellular vesicles (BEVs) produced by the human commensal gut microbe Bacteroides thetaiotaomicron contain multiple high-affinity vitamin B12 binding proteins suggesting that the vesicles play a role in micronutrient scavenging. Vitamin B12 belongs to the cobamide family of cofactors that regulate microbial communities through their limited bioavailability. We show that B. thetaiotaomicron derived BEVs bind a variety of cobamides and not only deliver them back to the parental bacterium but also sequester the micronutrient from competing bacteria. Additionally, Caco-2 cells, representing a model intestinal epithelial barrier, acquire cobamide-bound vesicles and traffic them to lysosomes, thereby mimicking the physiological cobalamin-specific intrinsic factor-mediated uptake process. Our findings identify a novel cobamide binding activity associated with BEVs with far-reaching implications for microbiota and host health.

Resonance Raman analysis of intracellular vitamin B12 analogs in methanogenic archaea.

Methanogenic archaea (methanogens) are microorganisms that can synthesize methane. They are found in diverse environments ranging from paddy fields to animal digestive tracts to deep-sea hydrothermal vents. Investigating their distribution and physiological activity is crucial for the detailed analysis of the dynamics of greenhouse gas generation and the search for the environmental limits of life. In methanogens, cobamide cofactors (vitamin B12 analogs) play a key role in methane synthesis and carbon fixation, thus serving as a marker compound that metabolically characterizes them. Here, we report on resonance Raman detection of cobamides in methanogenic cells without destroying cells and provide structural insights into those cobamides. We succeeded in detecting cobamides in four representative methanogens Methanosarcina mazei, Methanosarcina barkeri, Methanopyrus kandleri, and Methanocaldococcus jannaschii. The former two are mesophilic, cytochrome-containing methanogens, whereas the latter two are hyperthermophilic, non-cytochrome-containing methanogens. The 532 nm-excited Raman spectra of single or multiple cells of the four species all showed resonance Raman bands of cobamides arising mainly from the corrin ring, with the most intense one at ∼1500 cm-1. We envision that resonance Raman microspectroscopy could be useful for in situ, nondestructive identification of methanogenic cells that produce high levels of cobamides.

Direct Cobamide Remodeling via Additional Function of Cobamide Biosynthesis Protein CobS from Vibrio cholerae.

Vitamin B12 belongs to a family of structurally diverse cofactors with over a dozen natural analogs, collectively referred to as cobamides. Most bacteria encode cobamide-dependent enzymes, many of which can only utilize a subset of cobamide analogs. Some bacteria employ a mechanism called cobamide remodeling, a process in which cobamides are converted into other analogs to ensure that compatible cobamides are available in the cell. Here, we characterize an additional pathway for cobamide remodeling that is distinct from the previously characterized ones. Cobamide synthase (CobS) is an enzyme required for cobamide biosynthesis that attaches the lower ligand moiety in which the base varies between analogs. In a heterologous model system, we previously showed that Vibrio cholerae CobS (VcCobS) unexpectedly conferred remodeling activity in addition to performing the known cobamide biosynthesis reaction. Here, we show that additional Vibrio species perform the same remodeling reaction, and we further characterize VcCobS-mediated remodeling using bacterial genetics and in vitro assays. We demonstrate that VcCobS acts upon the cobamide pseudocobalamin directly to remodel it, a mechanism which differs from the known remodeling pathways in which cobamides are first cleaved into biosynthetic intermediates. This suggests that some CobS homologs have the additional function of cobamide remodeling, and we propose the term "direct remodeling" for this process. This characterization of yet another pathway for remodeling suggests that cobamide profiles are highly dynamic in polymicrobial environments, with remodeling pathways conferring a competitive advantage. IMPORTANCE Cobamides are widespread cofactors that mediate metabolic interactions in complex microbial communities. Few studies directly examine cobamide profiles, but several have shown that mammalian gastrointestinal tracts are rich in cobamide analogs. Studies of intestinal bacteria, including beneficial commensals and pathogens, show variation in the ability to produce and utilize different cobamides. Some bacteria can convert imported cobamides into compatible analogs in a process called remodeling. Recent discoveries of additional cobamide remodeling pathways, including this work, suggest that remodeling is an important factor in cobamide dynamics. Characterization of such pathways is critical in understanding cobamide flux and nutrient cross-feeding in polymicrobial communities, and it facilitates the establishment of microbiome manipulation strategies via modulation of cobamide profiles.

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.

Structural and functional analysis of an l-serine O-phosphate decarboxylase involved in norcobamide biosynthesis.

Structural diversity of natural cobamides (Cbas, B12 vitamers) is limited to the nucleotide loop. The loop is connected to the cobalt-containing corrin ring via an (R)-1-aminopropan-2-ol O-2-phosphate (AP-P) linker moiety. AP-P is produced by the l-threonine O-3-phosphate (l-Thr-P) decarboxylase CobD. Here, the CobD homolog SMUL_1544 of the organohalide-respiring epsilonproteobacterium Sulfurospirillum multivorans was characterized as a decarboxylase that produces ethanolamine O-phosphate (EA-P) from l-serine O-phosphate (l-Ser-P). EA-P is assumed to serve as precursor of the linker moiety of norcobamides that function as cofactors in the respiratory reductive dehalogenase. SMUL_1544 (SmCobD) is a pyridoxal-5'-phosphate (PLP)-containing enzyme. The structural analysis of the SmCobD apoprotein combined with the characterization of truncated mutant proteins uncovered a role of the SmCobD N-terminus in efficient l-Ser-P conversion.

Curative effect and mechanism ofαlipoic acid combined with epalrestat and methylcobalamin in the treatment of type 2 diabetic peripheral neuropathy / 中国医师杂志

Objective To investigate the curative effect and mechanism of α lipoic acid combined with epalrestat and methylcobalamin in patients with type 2 diabetes mellitus (T2DM) complicated with pe-ripheral neuropathy ( DPN) . Methods A total of 160 cases of patients with DPN were randomly divided into the control group ( treated with methylcobalamin and epalrestat) and the observation group ( treated with methylcobalamin, epalrestat and α lipoic acid) , and all patients were treated for 4 weeks. The therapeutic effect, nerve conduction velocity, oxidative stress index and related proteins expression in serum were ob-served in two groups. Results The effective rate of treatment in the observation group was higher than that in the control group (87. 50％ vs 75. 0％) (χ2 = 4. 103,P<0. 05). After treatment, the sensory nerve conduction velocity ( SNCV) and motor nerve conduction velocity ( MNCV) of median nerve and common peroneal nerve were significantly better in the observation group than the control group ( P<0. 05 ) . After treatment, the level of superoxide dismutase ( SOD) was significantly higher in the observation group than the control group, while the level of malondialdehyde ( MDA ) was significantly lower the control group (P<0. 05). After treatment, the level of Bcl-2 protein was significantly higher in observation group than the control group, while the levels of Bax and Caspase-3 proteins were significantly lower than the control group (P<0. 05). Conclusions The application ofαlipoic acid combined to epalrestat and methylcobal-amin in the treatment of DPN can significantly improve the sensory and motor nerve conduction.

Efficacy of mecobalamine in the treatment of acute cerebral infarction combined with hyperhomocysteinemia and post-stroke depression / 中华老年医学杂志

Objective To study the effect of mecobalamine treatment on acute cerebral infarction combined with high homocysteine (Hcy) and post-stroke depression.Methods A total of 160 cerebral infarction patients with high Hcy were collected from January 2010 to December 2011 in our hospital.Patients were randomly divided into two groups:the control group (n=80,with routine therapy) and the treatment group (n=80,with routine therapy plus intramuscular injection with folic acid 500μg for 8 weeks).The plasma concentration of Hcy was determined by fluoresene polarization immunoassay.The improvement of depression,anxiety and life skills in patients were assessed by selfmade questionnaires and self-reporting inventory (SLC 90).Results There were no significant differences in plasma Hcy level in control group before versus after treatment [(21.86±4.76) μmol/L vs.(18.67±3.81) μmol/L,t=0.40,P＞0.05].Compared with before treatment,plasma Hcy level in treatment group was significantly reduced 4 weeks after treatment [(11.66 ± 3.28) μmol/L vs.(22.68±5.16) μmol/L,t=2.55,P＜0.01].There was a significant difference in plasma Hcy level between the treatment group and the control group after treatment [(11.66 ±3.28) μmol/L vs.(18.67±3.81) μmol/L,t=7.40,P＜0.01].TheSCL-90 total score,positive symptom items score,the total score and the average score of positive symptom items were significantly lower in treatment group after treatment than in treatment group before treatment (t=2.04,2.97,5.45,4.21,all P＜0.05) and in control group after treatment (t 7.40,7.58,12.78,4.15,all P＜0.05).The improvement on symptomatic patterns including somatization,obsessive-compulsive problems,interpersonal sensitivity,depression,anxiety and fear were better in treatment group after treatment than in treatment group before treatment (t 2.34,2.28,2.51,2.19,3.40,2.35,allP＜0.05) and in control group after treatment (t=4.10,5.04,5.12,4.18,5.48,5.20,all P＜0.05).There were no significant differences in the improvement in paranoid ideation between the treatment group before and after treatment (t=1.40,P＞0.05),between the treatment group and the control group after treatment (t=1.48,P＞0.05),and between the control group before and after treatment (P＞0.05).Conclusions Mecobalamine can effectively reduce plasma Hcy level in cerebral infarction patients with high plasma Hcy,and alleviate the symptoms of anxiety,depression,nervousness and other emotions.