scholarly journals De novo cobalamin biosynthesis, transport and assimilation and cobalamin-mediated regulation of methionine biosynthesis in Mycobacterium smegmatis

2020 ◽  
Author(s):  
Terry Kipkorir ◽  
Gabriel T. Mashabela ◽  
Timothy J. De Wet ◽  
Anastasia Koch ◽  
Lubbe Wiesner ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
De Novo ◽  
Time Lapse ◽  
Methionine Synthase ◽  
Vitamin B ◽  
Human Pathogen ◽  
Methionine Biosynthesis ◽  
Gene And Protein Expression ◽  
Cobalamin Biosynthesis

ABSTRACTCobalamin is an essential co-factor in all domains of life, yet its biosynthesis is restricted to some bacteria and archaea. Mycobacterium smegmatis, an environmental saprophyte frequently used as surrogate for the obligate human pathogen, M. tuberculosis, carries approximately 30 genes predicted to be involved in de novo cobalamin biosynthesis. M. smegmatis also encodes multiple cobalamin-dependent enzymes, including MetH, a methionine synthase which catalyses the final reaction in methionine biosynthesis. In addition to metH, M. smegmatis possesses a cobalamin-independent methionine synthase, metE, suggesting that enzyme selection – MetH or MetE – is regulated by cobalamin availability. Consistent with this notion, we previously described a cobalamin-sensing riboswitch controlling metE expression in M. tuberculosis. Here, we apply a targeted mass spectrometry-based approach to confirm de novo cobalamin biosynthesis in M. smegmatis during aerobic growth in vitro. We also demonstrate that M. smegmatis transports and assimilates exogenous cyanocobalamin (CNCbl; a.k.a. vitamin B12) and its precursor, dicyanocobinamide ((CN)2Cbi). Interestingly, the uptake of CNCbl and (CN)2Cbi appears restricted in M. smegmatis and dependent on the conditional essentiality of the cobalamin-dependent methionine synthase. Using gene and protein expression analyses combined with single-cell growth kinetics and live-cell time-lapse microscopy, we show that transcription and translation of metE are strongly attenuated by endogenous cobalamin. These results support the inference that metH essentiality in M. smegmatis results from riboswitch-mediated repression of MetE expression. Moreover, differences observed in cobalamin-dependent metabolism between M. smegmatis and M. tuberculosis provide some insight into the selective pressures which might have shaped mycobacterial metabolism for pathogenicity.IMPORTANCEAccumulating evidence suggests that alterations in cobalamin-dependent metabolism marked the evolution of Mycobacterium tuberculosis from an environmental ancestor to an obligate human pathogen. However, the roles of cobalamin in mycobacterial physiology and pathogenicity remain poorly understood. We used the non-pathogenic saprophyte, M. smegmatis, to investigate the production of cobalamin, transport and assimilation of cobalamin precursors, and the potential role of cobalamin in regulating methionine biosynthesis. We provide biochemical and genetic evidence confirming constitutive de novo cobalamin biosynthesis in M. smegmatis under standard laboratory conditions, in contrast with M. tuberculosis, which appears to lack de novo cobalamin biosynthetic capacity. We also demonstrate that the uptake of cyanocobalamin (vitamin B12) and its precursors is restricted in M. smegmatis, apparently depending on the need to service the co-factor requirements of the cobalamin-dependent methionine synthase. These observations support the utility of M. smegmatis as a model to elucidate key metabolic adaptations enabling mycobacterial pathogenicity.

scholarly journals De Novo Cobalamin Biosynthesis, Transport and Assimilation and Cobalamin-Mediated Regulation of Methionine Biosynthesis in Mycobacterium smegmatis

2021 ◽  
Author(s):  
Terry Kipkorir ◽  
Gabriel T. Mashabela ◽  
Timothy J. de Wet ◽  
Anastasia Koch ◽  
Lubbe Wiesner ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
De Novo ◽  
Time Lapse ◽  
Methionine Synthase ◽  
Vitamin B ◽  
Human Pathogen ◽  
Methionine Biosynthesis ◽  
Gene And Protein Expression ◽  
Cobalamin Biosynthesis

Cobalamin is an essential co-factor in all domains of life, yet its biosynthesis is restricted to some bacteria and archaea. Mycobacterium smegmatis, an environmental saprophyte frequently used as surrogate for the obligate human pathogen, M. tuberculosis, carries approximately 30 genes predicted to be involved in de novo cobalamin biosynthesis. M. smegmatis also encodes multiple cobalamin-dependent enzymes, including MetH, a methionine synthase which catalyzes the final reaction in methionine biosynthesis. In addition to metH, M. smegmatis possesses a cobalamin-independent methionine synthase, metE, suggesting that enzyme use – MetH or MetE – is regulated by cobalamin availability. Consistent with this notion, we previously described a cobalamin-sensing riboswitch controlling metE expression in M. tuberculosis. Here, we apply a targeted mass spectrometry-based approach to confirm de novo cobalamin biosynthesis in M. smegmatis during aerobic growth in vitro. We also demonstrate that M. smegmatis can transport and assimilate exogenous cyanocobalamin (CNCbl; a.k.a. vitamin B12) and its precursor, dicyanocobinamide ((CN)2Cbi). However, the uptake of CNCbl and (CN)2Cbi in this organism is restricted and seems dependent on the conditional essentiality of the cobalamin-dependent methionine synthase. Using gene and protein expression analyses combined with single-cell growth kinetics and live-cell time-lapse microscopy, we show that transcription and translation of metE are strongly attenuated by endogenous cobalamin. These results support the inference that metH essentiality in M. smegmatis results from riboswitch-mediated repression of MetE expression. Moreover, differences observed in cobalamin-dependent metabolism between M. smegmatis and M. tuberculosis provide some insight into the selective pressures which might have shaped mycobacterial metabolism for pathogenicity. IMPORTANCE Alterations in cobalamin-dependent metabolism have marked the evolution of Mycobacterium tuberculosis as human pathogen. However, the role(s) of cobalamin in mycobacterial physiology remain poorly understood. Using the non-pathogenic saprophyte, M. smegmatis, we investigated the production of cobalamin, transport and assimilation of cobalamin precursors, and the role of cobalamin in regulating methionine biosynthesis. We confirm constitutive de novo cobalamin biosynthesis in M. smegmatis, in contrast with M. tuberculosis, which appears to lack de novo cobalamin biosynthetic capacity. We also show that uptake of cyanocobalamin (vitamin B12) and its precursors is restricted in M. smegmatis, apparently depending on the co-factor requirements of the cobalamin-dependent methionine synthase. These observations establish M. smegmatis as informative foil to elucidate key metabolic adaptations enabling mycobacterial pathogenicity.

scholarly journals Monomeric NADH-Oxidizing Methylenetetrahydrofolate Reductases from Mycobacterium smegmatis Lack Flavin Coenzyme

2020 ◽  
Vol 202 (12) ◽  
Author(s):  
Shivjee Sah ◽  
Kuldeep Lahry ◽  
Chandana Talwar ◽  
Sudhir Singh ◽  
Umesh Varshney
Keyword(s):  
Mycobacterium Smegmatis ◽  
De Novo ◽  
Aminosalicylic Acid ◽  
Methionine Biosynthesis ◽  
Content Type ◽  
Folate Pathway ◽  
Essential Enzyme ◽  
Antifolate Resistance

ABSTRACT 5,10-Methylenetetrahydrofolate reductase (MetF/MTHFR) is an essential enzyme in one-carbon metabolism for de novo biosynthesis of methionine. Our in vivo and in vitro analyses of MSMEG_6664/MSMEI_6484, annotated as putative MTHFR in Mycobacterium smegmatis, failed to reveal their function as MTHFRs. However, we identified two hypothetical proteins, MSMEG_6596 and MSMEG_6649, as noncanonical MTHFRs in the bacterium. MTHFRs are known to be oligomeric flavoproteins. Both MSMEG_6596 and MSMEG_6649 are monomeric proteins and lack flavin coenzymes. In vitro, the catalytic efficiency (kcat/Km) of MSMEG_6596 (MTHFR1) for 5,10-CH2-THF and NADH was ∼13.5- and 15.3-fold higher than that of MSMEG_6649 (MTHFR2). Thus, MSMEG_6596 is the major MTHFR. This interpretation was further supported by better rescue of the E. coli Δmthfr strain by MTHFR1 than by MTHFR2. As identified by liquid chromatography-tandem mass spectrometry, the product of MTHFR1- or MTHFR2-catalyzed reactions was 5-CH3-THF. The M. smegmatis Δmsmeg_6596 strain was partially auxotrophic for methionine and grew only poorly without methionine or without being complemented with a functional copy of MTHFR1 or MTHFR2. Furthermore, the Δmsmeg_6596 strain was more sensitive to folate pathway inhibitors (sulfachloropyridazine, p-aminosalicylic acid, sulfamethoxazole, and trimethoprim). The studies reveal that MTHFR1 and MTHFR2 are two noncanonical MTHFR proteins that are monomeric and lack flavin coenzyme. Both MTHFR1 and MTHFR2 are involved in de novo methionine biosynthesis and required for antifolate resistance in mycobacteria. IMPORTANCE MTHFR/MetF is an essential enzyme in a one-carbon metabolic pathway for de novo biosynthesis of methionine. MTHFRs are known to be oligomeric flavoproteins. Our in vivo and in vitro analyses of Mycobacterium smegmatis MSMEG_6664/MSMEI_6484, annotated as putative MTHFR, failed to reveal their function as MTHFRs. However, we identified two of the hypothetical proteins, MSMEG_6596 and MSMEG_6649, as MTHFR1 and MTHFR2, respectively. Interestingly, both MTHFRs are monomeric and lack flavin coenzymes. M. smegmatis deleted for the major mthfr (mthfr1) was partially auxotroph for methionine and more sensitive to folate pathway inhibitors (sulfachloropyridazine, para-aminosalicylic acid, sulfamethoxazole, and trimethoprim). The studies reveal that MTHFR1 and MTHFR2 are novel MTHFRs involved in de novo methionine biosynthesis and required for antifolate resistance in mycobacteria.

scholarly journals Interrelations between Glycine Betaine Catabolism and Methionine Biosynthesis in Sinorhizobium meliloti Strain 102F34

2006 ◽  
Vol 188 (20) ◽  
pp. 7195-7204 ◽  
Author(s):  
Lise Barra ◽  
Catherine Fontenelle ◽  
Gwennola Ermel ◽  
Annie Trautwetter ◽  
Graham C. Walker ◽  
...  
Keyword(s):  
Glycine Betaine ◽  
Sinorhizobium Meliloti ◽  
Wild Type Strain ◽  
Methionine Synthase ◽  
Vitamin B ◽  
Wild Type ◽  
Methionine Biosynthesis ◽  
Catabolic Pathway ◽  
Methyl Transferase ◽  
Meliloti Strain

ABSTRACT Methionine is produced by methylation of homocysteine. Sinorhizobium meliloti 102F34 possesses only one methionine synthase, which catalyzes the transfer of a methyl group from methyl tetrahydrofolate to homocysteine. This vitamin B12-dependent enzyme is encoded by the metH gene. Glycine betaine can also serve as an alternative methyl donor for homocysteine. This reaction is catalyzed by betaine-homocysteine methyl transferase (BHMT), an enzyme that has been characterized in humans and rats. An S. meliloti gene whose product is related to the human BHMT enzyme has been identified and named bmt. This enzyme is closely related to mammalian BHMTs but has no homology with previously described bacterial betaine methyl transferases. Glycine betaine inhibits the growth of an S. meliloti bmt mutant in low- and high-osmotic strength media, an effect that correlates with a decrease in the catabolism of glycine betaine. This inhibition was not observed with other betaines, like homobetaine, dimethylsulfoniopropionate, and trigonelline. The addition of methionine to the growth medium allowed a bmt mutant to recover growth despite the presence of glycine betaine. Methionine also stimulated glycine betaine catabolism in a bmt strain, suggesting the existence of another catabolic pathway. Inactivation of metH or bmt did not affect the nodulation efficiency of the mutants in the 102F34 strain background. Nevertheless, a metH strain was severely defective in competing with the wild-type strain in a coinoculation experiment.

scholarly journals Complementation of Cobalamin Auxotrophy in Synechococcus sp. Strain PCC 7002 and Validation of a Putative Cobalamin RiboswitchIn Vivo

2016 ◽  
Vol 198 (19) ◽  
pp. 2743-2752 ◽  
Author(s):  
Adam A. Pérez ◽  
Zhenfeng Liu ◽  
Dmitry A. Rodionov ◽  
Zhongkui Li ◽  
Donald A. Bryant
Keyword(s):  
Large Scale ◽  
De Novo ◽  
Expression System ◽  
Industrial Applications ◽  
Methionine Synthase ◽  
Methionine Biosynthesis ◽  
Content Type ◽  
Methyl Donor ◽  
Synechococcus Sp

ABSTRACTThe euryhaline cyanobacteriumSynechococcussp. strain PCC 7002 has an obligate requirement for exogenous vitamin B12(cobalamin), but little is known about the roles of this compound in cyanobacteria. Bioinformatic analyses suggest that only the terminal enzyme in methionine biosynthesis, methionine synthase, requires cobalamin as a coenzyme inSynechococcussp. strain PCC 7002. Methionine synthase (MetH) catalyzes the transfer of a methyl group fromN5-methyl-5,6,7,8-tetrahydrofolate tol-homocysteine duringl-methionine synthesis and uses methylcobalamin as an intermediate methyl donor. Numerous bacteria and plants alternatively employ a cobalamin-independent methionine synthase isozyme, MetE, that catalyzes the same methyl transfer reaction as MetH but usesN5-methyl-5,6,7,8-tetrahydrofolate directly as the methyl donor. The cobalamin auxotrophy ofSynechococcussp. strain PCC 7002 was complemented by using themetEgene from the closely related cyanobacteriumSynechococcussp. strain PCC 73109, which possesses genes for both methionine synthases. This result suggests that methionine biosynthesis is probably the sole use of cobalamin inSynechococcussp. strain PCC 7002. Furthermore, a cobalamin-repressible gene expression system was developed inSynechococcussp. strain PCC 7002 that was used to validate the presence of a cobalamin riboswitch in the promoter region ofmetEfromSynechococcussp. strain PCC 73109. This riboswitch acts as a cobalamin-dependent transcriptional attenuator formetEin that organism.IMPORTANCESynechococcussp. strain PCC 7002 is a cobalamin auxotroph because, like eukaryotic marine algae, it uses a cobalamin-dependent methionine synthase (MetH) for the final step ofl-methionine biosynthesis but cannot synthesize cobalaminde novo. Heterologous expression ofmetE, encoding cobalamin-independent methionine synthase, fromSynechococcussp. strain PCC 73109, relieved this auxotrophy and enabled the construction of a truly autotrophicSynechococcussp. strain PCC 7002 more suitable for large-scale industrial applications. Characterization of a cobalamin riboswitch expands the genetic toolbox forSynechococcussp. strain PCC 7002 by providing a cobalamin-repressible expression system.

scholarly journals Inhibiting Pyridoxal Kinase of Entamoeba histolytica Is Lethal for This Pathogen

2021 ◽  
Vol 11 ◽  
Author(s):  
Suneeta Devi ◽  
Priya Tomar ◽  
Khaja Faisal Tarique ◽  
Samudrala Gourinath
Keyword(s):  
Entamoeba Histolytica ◽  
Drug Targets ◽  
Cultured Cells ◽  
De Novo ◽  
Active Form ◽  
Protozoan Parasite ◽  
Vitamin B ◽  
Pyridoxal Kinase ◽  
Small Molecule Libraries

Pyridoxal 5’-phosphate (PLP) functions as a cofactor for hundreds of different enzymes that are crucial to the survival of microorganisms. PLP-dependent enzymes have been extensively characterized and proposed as drug targets in Entamoeba histolytica. This pathogen is unable to synthesize vitamin B6via de-novo pathway and relies on the uptake of vitamin B6 vitamers from the host which are then phosphorylated by the enzyme pyridoxal kinase to produce PLP, the active form of vitamin B6. Previous studies from our lab shows that EhPLK is essential for the survival and growth of this protozoan parasite and its active site differs significantly with respect to its human homologue making it a potential drug target. In-silico screening of EhPLK against small molecule libraries were performed and top five ranked molecules were shortlisted on the basis of docking scores. These compounds dock into the PLP binding site of the enzyme such that binding of these compounds hinders the binding of substrate. Of these five compounds, two compounds showed inhibitory activity with IC50 values between 100-250 μM when tested in-vitro. The effect of these compounds proved to be extremely lethal for Entamoeba trophozoites in cultured cells as the growth was hampered by 91.5% and 89.5% when grown in the presence of these compounds over the period of 72 hours.

scholarly journals Flexible Cobamide Metabolism in Clostridioides (Clostridium) difficile 630 Δerm

2019 ◽  
Vol 202 (2) ◽  
Author(s):  
Amanda N. Shelton ◽  
Xun Lyu ◽  
Michiko E. Taga
Keyword(s):  
De Novo ◽  
Aminolevulinic Acid ◽  
Genomic Analysis ◽  
Opportunistic Pathogen ◽  
Vitamin B ◽  
Uptake System ◽  
Human Gut ◽  
Content Type ◽  
Clostridioides Difficile

ABSTRACT Clostridioides (Clostridium) difficile is an opportunistic pathogen known for its ability to colonize the human gut under conditions of dysbiosis. Several aspects of its carbon and amino acid metabolism have been investigated, but its cobamide (vitamin B12 and related cofactors) metabolism remains largely unexplored. C. difficile has seven predicted cobamide-dependent pathways encoded in its genome in addition to a nearly complete cobamide biosynthesis pathway and a cobamide uptake system. To address the importance of cobamides to C. difficile, we studied C. difficile 630 Δerm and mutant derivatives under cobamide-dependent conditions in vitro. Our results show that C. difficile can use a surprisingly diverse array of cobamides for methionine and deoxyribonucleotide synthesis and can use alternative metabolites or enzymes, respectively, to bypass these cobamide-dependent processes. C. difficile 630 Δerm produces the cobamide pseudocobalamin when provided the early precursor 5-aminolevulinic acid or the late intermediate cobinamide (Cbi) and produces other cobamides if provided an alternative lower ligand. The ability of C. difficile 630 Δerm to take up cobamides and Cbi at micromolar or lower concentrations requires the transporter BtuFCD. Genomic analysis revealed genetic variations in the btuFCD loci of different C. difficile strains, which may result in differences in the ability to take up cobamides and Cbi. These results together demonstrate that, like other aspects of its physiology, cobamide metabolism in C. difficile is versatile. IMPORTANCE The ability of the opportunistic pathogen Clostridioides difficile to cause disease is closely linked to its propensity to adapt to conditions created by dysbiosis of the human gut microbiota. The cobamide (vitamin B12) metabolism of C. difficile has been underexplored, although it has seven metabolic pathways that are predicted to require cobamide-dependent enzymes. Here, we show that C. difficile cobamide metabolism is versatile, as it can use a surprisingly wide variety of cobamides and has alternative functions that can bypass some of its cobamide requirements. Furthermore, C. difficile does not synthesize cobamides de novo but produces them when given cobamide precursors. A better understanding of C. difficile cobamide metabolism may lead to new strategies to treat and prevent C. difficile-associated disease.

scholarly journals The role of Saccharomyces cerevisiae Met1p and Met8p in sirohaem and cobalamin biosynthesis

1999 ◽  
Vol 338 (3) ◽  
pp. 701-708 ◽  
Author(s):  
Evelyne RAUX ◽  
Treasa McVEIGH ◽  
Sarah E. PETERS ◽  
Thomas LEUSTEK ◽  
Martin J. WARREN
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Biosynthetic Pathways ◽  
Subtle Difference ◽  
Pseudomonas Denitrificans ◽  
E Coli ◽  
His Tag ◽  
Cobalamin Biosynthesis

MET1 and MET8 mutants of Saccharomyces cerevisiae can be complemented by Salmonella typhimurium cysG, indicating that the genes are involved in the transformation of uroporphyrinogen III into sirohaem. In the present study, we have demonstrated complementation of defined cysG mutants of Sal. typhimurium and Escherichia coli, with either MET1 or MET8 cloned in tandem with Pseudomonas denitrificans cobA. The conclusion drawn from these experiments is that MET1 encodes the S-adenosyl-l-methionine uroporphyrinogen III transmethylase activity, and MET8 encodes the dehydrogenase and chelatase activities (all three functions are encoded by Sal. typhimurium and E. coli cysG). MET8 was further cloned into pET14b to allow expression of the protein with an N-terminal His-tag. After purification, the functions of the His-tagged Met8p were studied in vitro by assay with precorrin-2 in the presence of NAD+ and Co2+. The results demonstrated that Met8p acts as a dehydrogenase and chelatase in the biosynthesis of sirohaem. Moreover, despite the fact that S. cerevisiae does not make cobalamins de novo, we have shown also that MET8 is able to complement cobalamin cobaltochelatase mutants and have revealed a subtle difference in the early stages of the anaerobic cobalamin biosynthetic pathways between Sal. typhimurium and Bacillus megaterium.

scholarly journals Thermotoga lettingae Can Salvage Cobinamide To Synthesize Vitamin B12

2013 ◽  
Vol 79 (22) ◽  
pp. 7006-7012 ◽  
Author(s):  
Nicholas C. Butzin ◽  
Michael A. Secinaro ◽  
Kristen S. Swithers ◽  
J. Peter Gogarten ◽  
Kenneth M. Noll
Keyword(s):  
Gene Cluster ◽  
Common Ancestor ◽  
De Novo ◽  
Recent Common Ancestor ◽  
Vitamin B ◽  
Ancestral State ◽  
Salvage Pathway ◽  
Content Type ◽  
Most Recent Common Ancestor

ABSTRACTWe recently reported that theThermotogalesacquired the ability to synthesize vitamin B12by acquisition of genes from two distantly related lineages,ArchaeaandFirmicutes(K. S. Swithers et al., Genome Biol. Evol. 4:730–739, 2012). Ancestral state reconstruction suggested that the cobinamide salvage gene cluster was present in theThermotogales' most recent common ancestor. We also predicted thatThermotoga lettingaecould not synthesize B12de novobut could use the cobinamide salvage pathway to synthesize B12. In this study, these hypotheses were tested, and we found thatTt. lettingaedid not synthesize B12de novobut salvaged cobinamide. The growth rate ofTt. lettingaeincreased with the addition of B12or cobinamide to its medium. It synthesized B12when the medium was supplemented with cobinamide, and no B12was detected in cells grown on cobinamide-deficient medium. Upstream of the cobinamide salvage genes is a putative B12riboswitch. In other organisms, B12riboswitches allow for higher transcriptional activity in the absence of B12. WhenTt. lettingaewas grown with no B12, the salvage genes were upregulated compared to cells grown with B12or cobinamide. Another gene cluster with a putative B12riboswitch upstream is thebtuFCDABC transporter, and it showed a transcription pattern similar to that of the cobinamide salvage genes. The BtuF proteins from species that can and cannot salvage cobinamides were shownin vitroto bind both B12and cobinamide. These results suggest thatThermotogalesspecies can use the BtuFCD transporter to import both B12and cobinamide, even if they cannot salvage cobinamide.

scholarly journals Two distinct pools of B12analogs reveal community interdependencies in the ocean

2016 ◽  
Vol 114 (2) ◽  
pp. 364-369 ◽  
Author(s):  
Katherine R. Heal ◽  
Wei Qin ◽  
Francois Ribalet ◽  
Anthony D. Bertagnolli ◽  
Willow Coyote-Maestas ◽  
...  
Keyword(s):  
De Novo ◽  
Heterotrophic Bacteria ◽  
Thalassiosira Pseudonana ◽  
Vitamin B ◽  
Surface Ocean ◽  
Cobalamin Biosynthesis ◽  
Genomic Analyses ◽  
Domains Of Life ◽  
And Function ◽  
Related Compounds

Organisms within all domains of life require the cofactor cobalamin (vitamin B12), which is produced only by a subset of bacteria and archaea. On the basis of genomic analyses, cobalamin biosynthesis in marine systems has been inferred in three main groups: select heterotrophic Proteobacteria, chemoautotrophic Thaumarchaeota, and photoautotrophic Cyanobacteria. Culture work demonstrates that many Cyanobacteria do not synthesize cobalamin but rather produce pseudocobalamin, challenging the connection between the occurrence of cobalamin biosynthesis genes and production of the compound in marine ecosystems. Here we show that cobalamin and pseudocobalamin coexist in the surface ocean, have distinct microbial sources, and support different enzymatic demands. Even in the presence of cobalamin, Cyanobacteria synthesize pseudocobalamin—likely reflecting their retention of an oxygen-independent pathway to produce pseudocobalamin, which is used as a cofactor in their specialized methionine synthase (MetH). This contrasts a model diatom,Thalassiosira pseudonana, which transported pseudocobalamin into the cell but was unable to use pseudocobalamin in its homolog of MetH. Our genomic and culture analyses showed that marine Thaumarchaeota and select heterotrophic bacteria produce cobalamin. This indicates that cobalamin in the surface ocean is a result of de novo synthesis by heterotrophic bacteria or via modification of closely related compounds like cyanobacterially produced pseudocobalamin. Deeper in the water column, our study implicates Thaumarchaeota as major producers of cobalamin based on genomic potential, cobalamin cell quotas, and abundance. Together, these findings establish the distinctive roles played by abundant prokaryotes in cobalamin-based microbial interdependencies that sustain community structure and function in the ocean.

scholarly journals Flexible cobamide metabolism in Clostridioides (Clostridium) difficile 630 Δerm

2019 ◽  
Author(s):  
Amanda N. Shelton ◽  
Xun Lyu ◽  
Michiko E. Taga
Keyword(s):  
Clostridium Difficile ◽  
De Novo ◽  
Genomic Analysis ◽  
Opportunistic Pathogen ◽  
Vitamin B ◽  
Uptake System ◽  
Human Gut ◽  
Clostridioides Difficile ◽  
Lower Ligand

AbstractClostridioides (Clostridium) difficile is an opportunistic pathogen known for its ability to colonize the human gut under conditions of dysbiosis. Several aspects of its carbon and amino acid metabolism have been investigated, but its cobamide (vitamin B12 and related cofactors) metabolism remains largely unexplored. C. difficile has seven predicted cobamide-dependent metabolisms encoded in its genome in addition to a nearly complete cobamide biosynthesis pathway and a cobamide uptake system. To address the importance of cobamides to C. difficile, we studied C. difficile 630 Δerm and mutant derivatives under cobamide-dependent conditions in vitro. Our results show that C. difficile can use a surprisingly diverse array of cobamides for methionine and deoxyribonucleotide synthesis, and can use alternative metabolites or enzymes, respectively, to bypass these cobamide-dependent processes. C. difficile 630 Δerm produces the cobamide pseudocobalamin when provided the early precursor 5-aminolevulinc acid or the late intermediate cobinamide, and produces other cobamides if provided an alternative lower ligand. The ability of C. difficile 630 Δerm to take up cobamides and Cbi at micromolar or lower concentrations requires the transporter BtuFCD. Genomic analysis revealed genetic variations in in the btuFCD locus of different C. difficile strains, which may result in differences in the ability to take up cobamides and Cbi. These results together demonstrate that, like other aspects of its physiology, cobamide metabolism in C. difficile is versatile.ImportanceThe ability of the opportunistic pathogen Clostridioides difficile to cause disease is closely linked to its propensity to adapt to conditions created by dysbiosis of the human gut microbiota. The cobamide (vitamin B12) metabolism of C. difficile has been underexplored, though it has seven metabolic pathways that are predicted to require cobamide-dependent enzymes. Here, we show that C. difficile cobamide metabolism is versatile, as it can use a surprisingly wide variety of cobamides and has alternative functions that can bypass some of its cobamide requirements. Furthermore, C. difficile does not synthesize cobamides de novo, but produces them when given cobamide precursors. Better understanding of C. difficile cobamide metabolism may lead to new strategies to treat and prevent C. difficile-associated disease.

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