Biology subjects

Mok, K. C.

Publications and source records attributed to Mok, K. C..

9 recordsLinked to original sources

bioRxiv2025

Cobamide-based interactions between soil bacteria can be predicted based on monoculture growth

Interactions between microbes shape the structure and function of microbial communities. While studying interactions is key to understanding microbial communities as a whole, gaining a detailed mechanistic view is challenging due to the number of co-occurring interactions. Here, we focus on a single class of shared nutrients, the cobalamin (vitamin B12) family of enzyme cofactors known as cobamides, to study nutrient interactions in laboratory consortia of increasing complexity. Measuring cobamide-dependent growth of two soil Caulobacter isolates in monoculture revealed that each grows faster at a distinct range of cobamide concentrations. Co-culturing the two isolates demonstrated that the cobamide concentration predictably determines which isolate predominates. These results suggest that, while the two organisms appear to be functionally redundant, with identical metabolic capabilities predicted in their genomes, they may occupy distinct niches in their environment based on cobamide concentration. We further examined the ability of cobamide-producing Mesorhizobium and Priestia isolates to share cobamides and found that both support the cobamide-dependent growth of the two Caulobacter isolates. The Caulobacter isolates could coexist in tricultures with each producer following serial passaging, indicating the producers can support the growth of multiple cobamide-dependent organisms. We analyzed the metabolic capacity encoded in the genomes of the four isolates and found that cobamides are likely the main shared nutrient in our co- and tri-cultures. These results highlight the utility of cobamides as a model nutrient to characterize and predict interactions in bacterial consortia of increasing complexity.

microbiology↗

bioRxiv2024

Microbes display broad diversity in cobamide preferences

Cobamides, the vitamin B12 (cobalamin) family of cofactors, are used by most organisms but produced by only a fraction of prokaryotes, and are thus considered key shared nutrients among microbes. Cobamides are structurally diverse, with multiple different cobamides found in most microbial communities. The ability to use different cobamides has been tested for several bacteria and microalgae, and nearly all show preferences for certain cobamides. This approach is limited by the commercial unavailability of cobamides other than cobalamin. Here, we have extracted and purified seven commercially unavailable cobamides to characterize bacterial cobamide preferences based on growth in specific cobamide-dependent conditions. The tested bacteria include engineered strains of Escherichia coli, Sinorhizobium meliloti, and Bacillus subtilis expressing native or heterologous cobamide-dependent enzymes, cultured under conditions that functionally isolate specific cobamide-dependent processes such as methionine synthesis. Comparison of these results to previous studies of diverse bacteria and microalgae revealed that a broad diversity of cobamide preferences exists not only across different organisms, but also between different cobamide-dependent metabolic pathways within the same organism. The microbes differed in the cobamides that support growth most efficiently, cobamides that do not support growth, and the minimum cobamide concentrations required for growth. The latter differ by up to four orders of magnitude across organisms from different environments and by up to 20-fold between cobamide-dependent enzymes within the same organism. Given that cobamides are shared, required for use of specific growth substrates, and essential for central metabolism in certain organisms, cobamide preferences likely impact community structure and function.

microbiology↗

bioRxiv2024

Vitamin B12 variants structure soil microbial communities despite soil's vast reservoir of B12.

Soil microbial communities perform critical ecosystem services through the collective metabolic activities of numerous individual organisms. Most microbes use corrinoids, a structurally diverse family of cofactors related to vitamin B12. Corrinoid structure influences the growth of individual microbes, yet how these growth responses scale to the community level remains unknown. Analysis of metagenome-assembled genomes suggests corrinoids are supplied to the community by members of the archaeal and bacterial phyla Thermoproteota, Actinobacteria, and Proteobacteria. Corrinoids were found largely adhered to the soil matrix in a grassland soil, at levels exceeding those required by cultured bacteria. Enrichment cultures and soil microcosms seeded with different corrinoids showed distinct shifts in bacterial community composition, supporting the hypothesis that corrinoid structure can shape communities. Environmental context influenced both community and taxon-specific responses to specific corrinoids. These results implicate corrinoids as key determinants of soil microbiome structure and suggest that environmental micronutrient reservoirs promote community stability.

microbiology↗

bioRxiv2024

Laboratory evolution of E. coli with a natural vitamin B12 analog reveals roles for cobamide uptake and adenosylation in methionine synthase-dependent growth

Bacteria encounter chemically similar nutrients in their environment that impact their growth in distinct ways. Among such nutrients are cobamides, the structurally diverse family of cofactors related to vitamin B12 (cobalamin), which function as cofactors for diverse metabolic processes. Given that different environments contain varying abundances of different cobamides, bacteria are likely to encounter cobamides that enable them to grow robustly as well as those that do not function efficiently for their metabolism. Here, we performed a laboratory evolution of a cobamide-dependent strain of Escherichia coli with pseudocobalamin (pCbl), a cobamide that E. coli uses less effectively than cobalamin for MetH-dependent methionine synthesis, to identify genetic adaptations that lead to improved growth with less-preferred cobamides. After propagating and sequencing nine independent lines and validating the results by constructing targeted mutations, we found that mutations that increase expression of the outer membrane cobamide transporter BtuB are beneficial during growth under cobamide-limiting conditions. Unexpectedly, we also found that overexpression of the cobamide adenosyltransferase BtuR confers a specific growth advantage in pCbl. Characterization of the latter phenotype revealed that BtuR and adenosylated cobamides contribute to optimal MetH-dependent growth. Together, these findings improve our understanding of how bacteria expand their cobamide-dependent metabolic potential. ImportanceIn nature, bacteria commonly experience fluctuations in the availability of required nutrients. Thus, their environment often contains nutrients that are insufficient in quantity or that function poorly in their metabolism. Cobamides, the vitamin B12 family of cofactors, are ideal for investigating the influence of nutrient quality on bacterial growth. We performed a laboratory evolution experiment in E. coli with a less-preferred cobamide to examine whether and how bacteria can improve their growth with less ideal nutrients. We found that overexpression of genes for cobamide uptake and modification are genetic adaptations that improve growth under these conditions. Given that cobamides are key shared metabolites in microbial communities, our results reveal insights into bacterial interactions and competition for nutrients.

microbiology↗

bioRxiv2023

Phylogenetic distribution and experimental characterization of corrinoid production and dependence in soil bacterial isolates

Soil microbial communities impact carbon sequestration and release, biogeochemical cycling, and agricultural yields. These global effects rely on metabolic interactions that modulate community composition and function. However, the physicochemical and taxonomic complexity of soil and the scarcity of available isolates for phenotypic testing are significant barriers to studying soil microbial interactions. Corrinoids--the vitamin B12 family of cofactors--are critical for microbial metabolism, yet they are synthesized by only a subset of microbiome members. Here, we evaluated corrinoid production and dependence in soil bacteria as a model to investigate the ecological roles of microbes involved in metabolic interactions. We isolated and characterized a taxonomically diverse collection of 161 soil bacteria from a single study site. Most corrinoid-dependent bacteria in the collection prefer B12 over other corrinoids, while all tested producers synthesize B12, indicating metabolic compatibility between producers and dependents in the collection. Furthermore, a subset of producers release B12 at levels sufficient to support dependent isolates in laboratory culture at estimated ratios of up to 1,000 dependents per producer. Within our isolate collection, we did not find strong phylogenetic patterns in corrinoid production or dependence. Upon investigating trends in the phylogenetic dispersion of corrinoid metabolism categories across sequenced bacteria from various environments, we found that these traits are conserved in 47 out of 85 genera. Together, these phenotypic and genomic results provide evidence for corrinoid-based metabolic interactions among bacteria and provide a framework for the study of nutrient-sharing ecological interactions in microbial communities.

microbiology↗

bioRxiv2022

Cobalamin riboswitches are broadly sensitive to corrinoid cofactors to enable an efficient gene regulatory strategy

In bacteria, many essential metabolic processes are controlled by riboswitches, gene regulatory RNAs that directly bind and detect metabolites. Highly specific effector binding enables riboswitches to respond to a single biologically relevant metabolite. Cobalamin riboswitches are a potential exception because over a dozen chemically similar but functionally distinct cobalamin variants (corrinoid cofactors) exist in nature. Here, we measured cobalamin riboswitch activity in vivo using a Bacillus subtilis fluorescent reporter system and found that among 38 tested riboswitches, a subset responded to corrinoids promiscuously, while others were semi-selective. Analyses of chimeric riboswitches and structural models indicate that, unlike other riboswitch classes, cobalamin riboswitches indirectly differentiate among corrinoids by sensing differences in their structural conformation. This regulatory strategy aligns riboswitch-corrinoid specificity with cellular corrinoid requirements in a B. subtilis model. Thus, bacteria can employ broadly sensitive riboswitches to cope with the chemical diversity of essential metabolites.

microbiology↗

bioRxiv2020

Identification of a novel cobamide remodeling enzyme in the beneficial human gut bacterium Akkermansia muciniphila

The beneficial human gut bacterium Akkermansia muciniphila provides metabolites to other members of the gut microbiota by breaking down host mucin, but most of its other metabolic functions have not been investigated. A. muciniphila is known to use cobamides, the vitamin B12 family of cofactors with structural diversity in the lower ligand, though the specific cobamides it can use have not been examined. We found that growth of A. muciniphila strain MucT was nearly identical with each of seven cobamides tested, in contrast to nearly all bacteria that have been studied. Unexpectedly, this promiscuity is due to cobamide remodeling - the removal and replacement of the lower ligand - despite the absence of the canonical remodeling enzyme CbiZ in A. muciniphila. We identified a novel enzyme, CbiR, that is capable of initiating the remodeling process by hydrolyzing the phosphoribosyl bond in the nucleotide loop of cobamides. CbiR does not share homology with other cobamide remodeling enzymes or B12-binding domains, and instead is a member of the AP endonuclease 2 enzyme superfamily. We speculate that CbiR enables bacteria to repurpose cobamides they otherwise cannot use in order to grow under a cobamide-requiring condition; this function was confirmed by heterologous expression of cbiR in E. coli. Homologs of CbiR are found in over 200 microbial taxa across 22 phyla, suggesting that many bacteria may use CbiR to gain access to the diverse cobamides present in their environment. ImportanceCobamides, the vitamin B12 family of cobalt-containing cofactors, are required for metabolism in all domains of life, including most bacteria. Cobamides have structural variability in the lower ligand, and selectivity for particular cobamides has been observed in most organisms studied to date. Here, we discover that the beneficial human gut bacterium Akkermansia muciniphila can use a diverse range of cobamides due to its ability to change the cobamide structure via "cobamide remodeling". We identify and characterize the novel enzyme CbiR that is necessary for initiating the cobamide remodeling process. The discovery of this enzyme has implications not only for understanding the ecological role of A. muciniphila in the gut, but for other bacteria that carry this enzyme as well.

microbiology↗

bioRxiv2019

Cofactor selectivity in methylmalonyl-CoA 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-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.

microbiology↗

bioRxiv2018

Uneven distribution of cobamide biosynthesis and dependence in bacteria predicted by comparative genomics

The vitamin B12 family of cofactors known as cobamides are essential for a variety of microbial metabolisms. We used comparative genomics of 11,000 bacterial species to analyze the extent and distribution of cobamide production and use across bacteria. We find that 86% of bacteria in this data set have at least one of 15 cobamide-dependent enzyme families, yet only 37% are predicted to synthesize cobamides de novo. The distribution of cobamide biosynthesis varies at the phylum level, with 57% of Actinobacteria, 45% of Proteobacteria, and 30% of Firmicutes, and less than 1% of Bacteroidetes containing the complete biosynthetic pathway. Cobamide structure could be predicted for 58% of cobamide-producing species, based on the presence of signature lower ligand biosynthesis and attachment genes. Our predictions also revealed that 17% of bacteria that have partial biosynthetic pathways, yet have the potential to salvage cobamide precursors. These include a newly defined, experimentally verified category of bacteria lacking the first step in the biosynthesis pathway. These predictions highlight the importance of cobamide and cobamide precursor crossfeeding as examples of nutritional dependencies in bacteria.

microbiology↗