RESUMO
Cobalamin (vitamin B12, herein referred to as B12) is an essential cofactor for most marine prokaryotes and eukaryotes1,2. Synthesized by a limited number of prokaryotes, its scarcity affects microbial interactions and community dynamics2-4. Here we show that two bacterial B12 auxotrophs can salvage different B12 building blocks and cooperate to synthesize B12. A Colwellia sp. synthesizes and releases the activated lower ligand α-ribazole, which is used by another B12 auxotroph, a Roseovarius sp., to produce the corrin ring and synthesize B12. Release of B12 by Roseovarius sp. happens only in co-culture with Colwellia sp. and only coincidently with the induction of a prophage encoded in Roseovarius sp. Subsequent growth of Colwellia sp. in these conditions may be due to the provision of B12 by lysed cells of Roseovarius sp. Further evidence is required to support a causative role for prophage induction in the release of B12. These complex microbial interactions of ligand cross-feeding and joint B12 biosynthesis seem to be widespread in marine pelagic ecosystems. In the western and northern tropical Atlantic Ocean, bacteria predicted to be capable of salvaging cobinamide and synthesizing only the activated lower ligand outnumber B12 producers. These findings add new players to our understanding of B12 supply to auxotrophic microorganisms in the ocean and possibly in other ecosystems.
Assuntos
Alteromonadaceae , Ligantes , Rhodobacteraceae , Vitamina B 12 , Oceano Atlântico , Técnicas de Cocultura , Interações Microbianas , Prófagos/genética , Prófagos/crescimento & desenvolvimento , Prófagos/metabolismo , Vitamina B 12/biossíntese , Vitamina B 12/química , Vitamina B 12/metabolismo , Alteromonadaceae/crescimento & desenvolvimento , Alteromonadaceae/metabolismo , Rhodobacteraceae/citologia , Rhodobacteraceae/metabolismo , Rhodobacteraceae/virologia , Ribonucleosídeos/metabolismo , Cobamidas/metabolismo , EcossistemaRESUMO
Small RNAs (sRNAs) and riboswitches represent distinct classes of RNA regulators that control gene expression upon sensing metabolic or environmental variations. While sRNAs and riboswitches regulate gene expression by affecting mRNA and protein levels, existing studies have been limited to the characterization of each regulatory system in isolation, suggesting that sRNAs and riboswitches target distinct mRNA populations. We report that the expression of btuB in Escherichia coli, which is regulated by an adenosylcobalamin (AdoCbl) riboswitch, is also controlled by the small RNAs OmrA and, to a lesser extent, OmrB. Strikingly, we find that the riboswitch and sRNAs reduce mRNA levels through distinct pathways. Our data show that while the riboswitch triggers Rho-dependent transcription termination, sRNAs rely on the degradosome to modulate mRNA levels. Importantly, OmrA pairs with the btuB mRNA through its central region, which is not conserved in OmrB, indicating that these two sRNAs may have specific targets in addition to their common regulon. In contrast to canonical sRNA regulation, we find that OmrA repression of btuB is lost using an mRNA binding-deficient Hfq variant. Together, our study demonstrates that riboswitch and sRNAs modulate btuB expression, providing an example of cis- and trans-acting RNA-based regulatory systems maintaining cellular homeostasis.
Assuntos
Cobamidas , Proteínas de Escherichia coli , Escherichia coli , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano , RNA Mensageiro , Riboswitch , Riboswitch/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Cobamidas/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Iniciação Traducional da Cadeia Peptídica , RNA Helicases/genética , RNA Helicases/metabolismo , Endorribonucleases/metabolismo , Endorribonucleases/genética , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Proteínas da Membrana Bacteriana Externa , Polirribonucleotídeo Nucleotidiltransferase , Proteínas de Membrana TransportadorasRESUMO
BtuM is a bacterial cobalamin transporter that binds the transported substrate in the base-off state, with a cysteine residue providing the α-axial coordination of the central cobalt ion via a sulfur-cobalt bond. Binding leads to decyanation of cobalamin variants with a cyano group as the ß-axial ligand. Here, we report the crystal structures of untagged BtuM bound to two variants of cobalamin, hydroxycobalamin and cyanocobalamin, and unveil the native residue responsible for the ß-axial coordination, His28. This coordination had previously been obscured by non-native histidines of His-tagged BtuM. A model in which BtuM initially binds cobinamide reversibly with low affinity (KD = 4.0 µM), followed by the formation of a covalent bond (rate constant of 0.163 s-1), fits the kinetics data of substrate binding and decyanation of the cobalamin precursor cobinamide by BtuM. The covalent binding mode suggests a mechanism not used by any other transport protein.
Assuntos
Proteínas de Bactérias , Modelos Moleculares , Ligação Proteica , Vitamina B 12 , Vitamina B 12/metabolismo , Vitamina B 12/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Sítios de Ligação , Cobalto/química , Cobalto/metabolismo , Cobamidas/metabolismo , Cobamidas/química , Cinética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Histidina/metabolismo , Histidina/químicaRESUMO
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 pathwayâisomerization of ß-l-lysine to erythro-3,5-diaminohexanoateâis 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.