Progress 10/01/14 to 09/30/19
Outputs
Target Audience:Microbiologists and chemical biologists from academic, government, and private settings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Laboratory personnel involved in the project were the following: Specialist: Kenny Mok Postdoctoral scholars: Sebastian Gude and Zachary Hallberg Graduate students: Olga Sokolovskaya, Amanda Shelton, Kristopher Kennedy, Gordon Pherribo, Alexa Nicolas Undergraduates: Xun Lyu, Soohan Woo, Victoria Innocent, Kim Kang Training activities included microbiology (all participants), analytical chemistry (all participants), bacterial genetics (Mok, Gude, Hallberg, Sokolovskaya, Shelton, Kennedy, Pherribo, Lyu, Kang), and writing manuscripts (Gude, Sokolovskaya, Shelton). Professional development activities included attending conferences and oral and poster presentations (Mok, Gude, Hallberg, Sokolovskaya, Shelton, Kennedy, Nicolas, Innocent). How have the results been disseminated to communities of interest?Activities have been disseminated to the scientific community through presentations at conferences (West Coast Bacterial Physiologists Meeting, Gordon Research Conferences, Microbiology Student Symposium, American Society for Microbiology Microbe conference). Microbiology-related activities were presented to the non-scientific community at the Berkeley Public Library. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Earth's soils, aquatic environments, and host-associated niches are teeming with microbial life. The microbes that inhabit these environments are too tiny to observe without the help of specialized tools, yet they have outsize impact on the ecosystems on which humans depend, including agriculture, industry, and native ecosystems. Climate change, pollution, and agricultural practices have put microbial communities in peril, disturbing the evolved balance of these ecosystems, with impacts rippling to their hosts and macroenvironments. This research provided foundational knowledge about the vitamin B12 family of cofactors (corrinoids) - microbial products that may be keystone metabolites in microbial communities that can be leveraged to improve the health of microbial communities and the environments they impact. In order to realize this potential application, an improved fundamental understanding of the roles of corrinoids in microbial physiology is necessary. This project focused on understanding how bacteria respond to the many distinct molecules that comprise the corrinoid family. By using the tools of biochemistry, molecular biology, and computational biology, we have pinpointed specific molecular features that endow bacteria with the ability to distinguish between the members of this important group of metabolites. Objective 1. Cobamides, corrinoid cofactors including vitamin B12, are 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. Previously, the biochemical basis for cobamide preferences was largely unknown. 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. The in vitro binding affinity of MCM for cobamides was 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. Cobamide-dependent growth of the plant root-nodulating 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. Objective 2. Bacteria were previously known to regulate the expression of genes involved in vitamin B12 uptake and biosynthesis by direct binding between B12 and noncoding RNA elements called riboswitches. Akin to corrinoid-dependent enzymes, the response of riboswitches to corrinoids other than B12 had largely not been studied. We hypothesized that corrinoid riboswitches - which are present in ~75% of human gut bacterial species - have evolved to respond selectively to corrinoids that benefit the organism's physiology. To determine the extent to which riboswitches from diverse bacteria can distinguish between structurally different corrinoids, we expressed 40 GFP reporter fusions to riboswitches from 10 bacterial species in a Bacillus subtilis strain that we engineered to import all corrinoids efficiently. Our results revealed that some riboswitches can indeed distinguish between corrinoids, while others responded similarly to all tested corrinoids. These results demonstrate that corrinoid riboswitches have higher functional diversity than was previously thought, suggesting that they have novel mechanisms of ligand selectivity and gene regulation. Objective 3. The majority of bacteria are unable to synthesize all of the metabolites they require, and must take up needed metabolites from the environment. We explored the question of how bacteria that produce corrinoids and other metabolites release these metabolites for uptake by other species, and how this process evolves. We established synthetic cocultures of engineered strains Escherichia coli to address these questions, using corrinoids and methionine as models to study metabolite release. We found that corrinoid structure influences the degree to which a corrinoid produced by one strain can support the growth of the other. In addition, we found that while methionine is not released by our wild type E. coli strain, it could be engineered to release methionine by increasing flux through the methionine biosynthetic pathway by several different strategies. This research could reveal not only the possible mechanisms of metabolite release, but also the mechanisms by which nutrient release can evolve. Objective 4. We conducted a comparative genomic study that enables us to predict cobamide biosynthesis and dependence across over 11,000 bacterial species. We found that 86% of bacteria in this data set have at least one of 15 cobamide-dependent enzyme families, but only 37% are predicted to synthesize cobamides de novo. The distribution of cobamide biosynthesis and use vary at the phylum level. While 57% of Actinobacteria are predicted to biosynthesize cobamides, only 0.6% of Bacteroidetes have the complete pathway, yet 96% of species in this phylum have cobamide-dependent enzymes. The form of cobamide produced by the bacteria 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 have partial biosynthetic pathways, yet have the potential to salvage cobamide precursors. Bacteria with a partial cobamide biosynthesis pathway include those in 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 salvaging as examples of nutritional dependencies in bacteria.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Sokolovskaya OM, Mok KC, Park JD, Tran JLA, Quanstrom KA, Taga ME. Cofactor selectivity in methylmalonyl coenzyme A mutase, a model cobamide-dependent enzyme. MBio 2019 10(5) pii: e01303-19.
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Microbiologists and chemical biologists from academic, government, and private settings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Laboratory personnel involved in the project were the following: Specialist: Kenny Mok Postdoctoral scholars: Sebastian Gude, Zachary Hallberg Graduate students: Olga Sokolovskaya, Amanda Shelton, Kristopher Kennedy, Gordon Pherribo, Alexa Nicolas Undergraduates: Anna Grimaldo, Joseph Maa Training activities included microbiology (all participants), analytical chemistry (Sokolovskaya, Shelton, Mok, Kennedy, Nicolas), bacterial genetics (Mok, Gude, Pherribo, Kennedy, Sokolovskaya, Maa), biochemistry (Sokolovskaya, Kennedy, Hallberg), bioinformatics (Nicolas, Shelton, Kennedy, Mok, Grimaldo), and scientific writing (all participants). Professional development activities included attending regional and/or international conferences (Mok, Sokolovskaya, Shelton, Gude, Kennedy, Pherribo, Nicolas, Maa), poster presentations (Sokolovskaya, Kennedy, Shelton), oral presentations (all participants), and career development conferences (Shelton). How have the results been disseminated to communities of interest?Results have been disseminated to the scientific community through presentations at conferences and invited seminars. Specifically, the PI has presented this research at the University of Florida, the University of California, San Francisco, the International Microbial Genomes Conference, the Gordon Research Conference on Tetrapyrroles, and the Gordon Research Conference on the Molecular Basis of Microbial One-Carbon Metabolism. Additionally, laboratory personnel have presented their research at venues including the West Coast Bacterial Physiologists conference, ABRCMS conference, Gordon Research Conferences, and the Berkeley Microbiology Retreat. What do you plan to do during the next reporting period to accomplish the goals? We will measure the corrinoid binding affinity and activity of orthologs of MCM, and complete the measurements of corrinoid use by S. meliloti, in order to generate a more complete understanding of the structure-function relationships between this model corrinoid-dependent enzyme and the cofactors it requires for its activity. We will continue to construct and measure activity of new riboswitch-GFP reporters in B. subtilis, directly measure gene expression from riboswitch-controlled operons in the native context, and examine the relationships between riboswitch length, sequence, specificity, and sensitivity, to generate a molecular-level understanding of regulation of gene expression by corrinoid riboswitch elements. We will continue to construct E. coli strains with alterations in methionine pathway flux, inducible prophages, and phage resistance, and test these strains in coculture with a methionine auxotroph to measure the effects of metabolic flux and cell lysis on nutrient sharing. We will establish additional cocultures to explore other nutrient crossfeeding relationships. We will continue our study of corrinoid biosynthesis and use in bacterial isolates in order to test and refine predictions of corrinoid biology in natural systems.
Impacts
What was accomplished under these goals?
We continued to examine the molecular specificity of corrinoid-enzyme and corrinoid-RNA interactions, and have further developed defined communities to investigate nutrient crossfeeding. Objective 1. We conducted biochemical experiments with a model corrinoid dependent enzyme, methylmalonyl-coenzyme A mutase from the bacterium Sinorhizobium meliloti (SmMCM). We have completed the measurement of the binding affinities of this enzyme for nine natural and five synthetic corrinoids. We found that this enzyme has highest affinity for the native corrinoid of S. meliloti but can bind several others with lower affinity. We do not observe binding at micromolar concentrations to corrinoids containing ring nitrogens in the lower axial ligand, even though a corrinoid lacking a lower ligand can bind. We found that the enzyme is active in vitro with all of the corrinoids for which we observed binding. We also measured the ability of corrinoids to support growth of a S. meliloti strain engineered to require corrinoids specifically for MCM activity and found that while all support growth, there is a 100-fold range in the effective concentrations. Objective 2. We constructed a Bacillus subtilis strain lacking its corrinoidp-binding proteins for heterologous expression of corrinoid riboswitch reporter constructs. Using this strain, we examined the corrinoid responsiveness of over 40 riboswitch-GFP reporters. These included both native riboswitches from a variety of bacteria, mostly from the Firmicutes phylum, as well as truncated or chimeric riboswitches designed to test the roles of particular riboswitch domains in regulatory function. We found that riboswitch-corrinoid specificity falls into two major categories based on their response to four representative corrinoids. This specificity is not encoded in only a single subdomain, as domain swapping experiments show complex changes in affinity and specificity. Objective 3. We developed a panel of synthetic cocultures containing engineered strains of E. coli. We focused on modulating the amount of methionine produced by one of the strains as a way to study the effect of metabolic flux on coculture dynamics. In cocultures containing two nutritional auxotrophs, the ratio between the two strains was altered based on the deliberate tuning of methioinine production levels. We have constructed a second panel of strains to test the effect of cell lysis on coculture dynamics. Objective 4. We completed and published a comprehensive comparative genomics analysis of corrinoid biology across bacteria. We found striking phylum-level differences in corrinoid production and use and identified and characterized a new class of corrinoid precursor auxotrophy. Based on these results, we are testing predictions for corrinoid biosynthesis and use in several natural bacterial isolates.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Hazra AB, Ballou DP, Taga ME. 2018. Unique Biochemical and Sequence Features Enable BluB To Destroy Flavin and Distinguish BluB from the Flavin Monooxygenase Superfamily. Biochemistry 57(11):1748-1757.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Shelton AN, Seth EC, Mok KC, Han AW, Jackson SN, Haft DR, Taga ME. 2018. Uneven distribution of cobamide biosynthesis and dependence in bacteria predicted by comparative genomics. ISME J. Nov 14. doi: 10.1038/s41396-018-0304-9. [Epub ahead of print]
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Microbiologists and chemical biologists from academic, government, and private settings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Laboratory personnel involved in the project were the following: Specialist: Kenny Mok Postdoctoral scholars: Nicole Abreu, Sebastian Gude Graduate students: Olga Sokolovskaya, Amanda Shelton, Kristopher Kennedy, Gordon Pherribo, Alexa Nicolas Undergraduates: Kathryn Quanstrom, Jenny Marino, Anna Grimaldo, Yue Clare Lou, Joseph Maa Training activities included microbiology (all participants), analytical chemistry (Sokolovskaya, Shelton, Pherribo, Grimaldo), bacterial genetics (Mok, Gude, Pherribo, Kennedy, Sokolovskaya, Quanstrom, Lou, Marino, Maa), biochemistry (Sokolovskaya, Kennedy), bioinformatics (Shelton, Kennedy, Mok, Grimaldo), and scientific writing (all participants). Professional development activities included attending regional and/or international conferences (Mok, Sokolovskaya, Shelton, Gude, Kennedy, Pherribo, Nicolas, Lou), poster presentations (Sokolovskaya, Kennedy, Pherribo, Shelton, Lou), oral presentations (all participants), and career development conferences (Shelton, Sokolovskaya, Pherribo). How have the results been disseminated to communities of interest?Results have been disseminated to the scientific community through presentations at conferences and invited seminars. Specifically, the PI has presented this research at the University of Tennessee, Marine Biological Laboratory, the Molecular Genetics of Bacteria and Phages Conference, NIH, and UC Berkeley. Additionally, laboratory personnel have presented their research at venues including the West Coast Bacterial Physiologists conference, ABRCMS conference, Gordon Research Conferences, and the Berkeley Microbiology Retreat. What do you plan to do during the next reporting period to accomplish the goals? Using sequence alignments and analysis of published X-ray crystal structures as a guide, we will design and test additional mutant forms of MCM for altered corrinoid binding profiles. These studies will allow us to dissect the sequence-function relationships in this enzyme family. We will develop high-throughput approaches to analyze riboswitch-corrinoid specificity in vivo using our established reporter assay. We will focus on understanding the modularity of riboswitch-based regulation in order to probe the mechanisms of riboswitch-corrinoid specificity. Additionally, we will perform in vitro binding and transcription assays and compare the results to the in vivo assays. Results will establish the relationship between corrinoid binding and gene regulation by different corrinoids. We will modify the E. coli coculture by making targeted deletion and overexpression constructs to modulate the biosynthesis and export of methionine. The coculture will be assayed for stability by passaging and measuring the abundance of each strain over time. We will establish additional cocultures to explore other nutrient crossfeeding relationships. We will continue our in vivo growth experiments to test the effect of corrinoid specificity on competition between bacteria and examine the evolution of corrinoid precursor auxotrophy.
Impacts
What was accomplished under these goals?
We are continuing to use the corrinoid model for microbial community interactions by examining the molecular specificity of corrinoid-enzyme and corrinoid-RNA interactions. Using this model, we are investigating corrinoid crossfeeding at the molecular level. Objective 1. We conducted biochemical experiments with a model corrinoid dependent enzyme, methylmalonyl-coenzyme A mutase from the bacterium Sinorhizobium meliloti (SmMCM). We measured the binding affinities of this enzyme for six natural and five synthetic corrinoids and measured the activity of the enzyme with three of these corrinoids. We additionally created mutant forms of the enzyme and tested the binding affinities to several corrinoids in vitro. Results showed that electrostatic interactions between the protein and ring atoms of the corrinoid lower ligand substantially influence binding affinity. We purified and tested two other bacterial MCM homologs and found that each has a distinct corrinoid specificity profile. Objective 2. We examined the corrinoid responsiveness of 50 riboswitch-GFP reporter constructs in an Escherichia coli strain with all genes encoding corrinoid-binding proteins deleted. Results showed that for only two of these constructs, reporter expression was controlled by specific corrinoids. The remaining constructs were introduced into a Bacillus subtilis reporter, where 43 showed corrinoid-dependent regulation. We investigated two pairs of naturally occurring tandem riboswitches and found that in both cases, each riboswitch had a unique corrinoid specificity profile, and when present in tandem upstream of the reporter gene, the corrinoid specificity profile was the sum of the individual riboswitches. These results demonstrate that (1) corrinoid riboswitches are capable of distinguishing between different corrinoids, and (2) corrinoid riboswitch-based regulation is modular. Objective 3. We developed a synthetic coculture containing two engineered strains of E. coli. One strain uses corrinoid precursors to produce cobalamin and supply it to a second strain, which uses cobalamin to produce methionine and provide it to the first strain. Preliminary results show that in coculture, both strains can grow, and growth is dependent on crossfeeding of cobalamin and methionine. However, the coculture does not persist when passaged into fresh medium because of an imbalance between abundant cobalamin production and limited methionine crossfeeding. Objective 4. We used results of our in vitro analysis of corrinoid specificity in MCM homologs (Objective 1) to design proof-of-concept experiments to test the effect of corrinoid specificity on bacterial competition. Using S. meliloti as a model, we constructed strains expressing MCM orthologs and mutants. The strains were competed in pairs in the presence of different corrinoids. These experiments are ongoing. We found striking phylum-level differences in corrinoid production and use and identified and characterized a new class of corrinoid precursor auxotrophy.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Xiaoji Liu, Barbara Turchi, Kenny C. Mok, Michiko E. Taga,
and Michael J. Miller (2017) HM2-phage resistant solventogenic Clostridium saccharoperbutylacetonicum N1-4 shows increased exopolysaccharide production. FEMS Microbiology Letters 364, 2017
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Microbiologists and chemical biologists from academic, government, and private settings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Laboratory personnel involved in the project were the following: Specialist: Kenny Mok Postdoctoral scholars: Amrita Hazra, Florian Widner, Nicole Abreu, Sebastian Gude Graduate students: Olga Sokolovskaya, Amanda Shelton, Kristopher Kennedy, Gordon Pherribo Undergraduates: Eshwar Manoharan, Jong Duk Park, Kathryn Quanstrom Training activities included microbiology (all participants), analytical chemistry (Sokolovskaya, Manoharan, Park, Hazra), bacterial genetics (Hazra, Mok, Pherribo, Kennedy, Quanstrom, Widner), biochemistry (Sokolovskaya, Park, Widner, Hazra), bioinformatics (Shelton, Hazra, Mok), and writing journal articles and grant applications (Abreu, Shelton). Professional development activities included attending regional and/or international conferences (Mok, Widner, Sokolovskaya, Shelton, Gude, Kennedy, Pherribo), poster presentations (Sokolovskaya, Mok, Widner, Kennedy, Pherribo, Shelton), oral presentations (all participants), and career development conferences (Abreu, Shelton). How have the results been disseminated to communities of interest?Results have been disseminated to the scientific community through presentations at conferences and invited seminars. Specifically, the PI has presented this research at the Max Planck Institute, Agilent Technologies, and UC Berkeley (for faculty as well as undergraduates in the Amgen Scholars Program). Additionally, laboratory personnel have presented their research at poster presentations at venues such as the Microbiology Retreat. What do you plan to do during the next reporting period to accomplish the goals? Binding and kinetic analysis of the four MCM homologs already partially characterized, as well as six more. In addition, we plan to construct and test mutant forms of MCM predicted to use different corrinoids, and test enzymes with unnatural corrinoid analogs in an effort to understand the sequence-function relationships in this enzyme family. Use our high-throughput in vivo riboswitch reporter assay to measure the corrinoid specificity of up to 100 additional riboswitches. We will use the specificity data generated in these experiments to predict and test sequence determinants of corrinoid specificity in riboswitches. Construct stable, nutrient-sharing cocultures of engineered bacteria and model their interactions.
Impacts
What was accomplished under these goals?
We are continuing to use the corrinoid model for microbial community interactions by examining the molecular specificity of corrinoid-enzyme and corrinoid-RNA interactions. Using this model, we are investigating corrinoid crossfeeding in synthetic and natural microbial communities. Objective 1. We recombinantly expressed and purified four bacterial methylmalonyl-coenzyme A mutase (MCM) homologs. We established biochemical assays to measure the binding affinity of these enzymes to corrinoids and to measure the activity of the enzyme. Preliminary work using these assays showed that all proteins are active with cobalamin (B12), and that the proteins have different affinities for different corrinoids. Objective 2. We made 40 riboswitch-RFP reporter constructs and tested their expression in E. coli in the presence and absence of cobalamin and another corrinoid, adeninylcobamide. One third of these constructs showed repression of RFP expression by one or both corrinoids. Based on this result, we have started to make second generation constructs containing additional riboswitches, with higher compatibility with E. coli. To minimize the effect of intracellular corrinoid-binding proteins on the riboswitch reporter results, we are constructing a strain containing deletions in all genes encoding corrinoid-binding proteins that are not essential for this assay. Objective 3. We developed a synthetic coculture containing engineered strains of E. coli and Sinorhizobium meliloti. Preliminary results show that in coculture, both strains can grow, and growth is dependent on crossfeeding of cobalamin and methionine. Objective 4. We used a termite model to examine the effect of manipulating corrinoid composition on the microbiome. Groups of termites were fed 0.05, 0.5, and 5 mM cresolylcobamide or cobinamide (a corrinoid with no lower ligand). To measure the effect of these treatments on community composition, we performed 16S amplicon sequencing on the termites from each group. The results showed potential differences in the different treatment regimens, but were largely inconclusive because of unexpected variability between termites originating from different mini-colonies.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Abreu NA, Taga ME (2016) Decoding molecular interactions in microbial communities. FEMS Microbiology Reviews 40(5):648-63
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Crespo-Rivas JC, Guefrachi I, Mok KC, Villa�cija-Aguilar JA, Acosta-Jurado S, Pierre O, Ruiz-Sainz JE, Taga ME, Mergaert P, Vinardell JM (2016) Sinorhizobium fredii HH103 bacteroids are not terminally differentiated and show altered O-antigen in nodules of the IRLC legume Glycyrrhiza uralensis. Environmental Microbiology 18:2392-404.
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Microbiologists and chemical biologists from academic, government, and private settings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Laboratory personnel involved in the project were the following: Specialist: Kenny Mok Postdoctoral scholars: Amrita Hazra, Florian Widner, Nicole Abreu Graduate students: Olga Sokolovskaya, Amanda Shelton Undergraduates: Vadim Osadchiy, Tristan Jordan, Elizabeth Lee, Eshwar Manoharan Training activities included microbiology (all participants), analytical chemistry (all participants), bacterial genetics (Mok, Osadchiy), writing grant applications (Abreu), and writing manuscripts (Mok, Hazra, Osadchiy). Professional development activities included attending regional and/or international conferences (Mok, Hazra, Widner, Abreu, Sokolovskaya, Shelton), poster presentations (Sokolovskaya, Mok, Widner, Jordan), and oral presentations (all participants). How have the results been disseminated to communities of interest?Activities have been disseminated to the scientific community through presentations at conferences and invited seminars. In addition, microbiology-related activities were presented to the non-scientific community at Cal Day, UC Berkeley's annual open house. What do you plan to do during the next reporting period to accomplish the goals? Biochemical analysis of MCM enzyme homologs from a larger number of bacteria: we will employ a coupled spectrophotometric assay to examine the kinetic parameters of MCM homologs from representative bacteria that produce different corrinoids. Analysis of corrinoid specificity of riboswitches: we will develop an in vivo reporter assay to examine the corrinoid specificity of a larger number of riboswitches with a panel of corrinoids. Develop defined model communities: we will predict nutritional interactions between bacteria by conducting a bioinformatic analysis of bacterial genomes, and subsequently test some of these predictions experimentally. Corrinoid manipulations in the termite gut model: we will examine the effect of adding complete corrinoids to the termite diet on corrinoid composition and community composition.
Impacts
What was accomplished under these goals?
Objective 1: We tested specificities of three corrinoid-dependent enzymes in the model bacterium Sinorhizobium meliloti. We functionally isolated activity of each enzyme by manipulating the genetic background and growth conditions. Growth with a range of concentrations of different purified corrinoids was measured in 96-well plates. We found that the concentration of each corrinoid required for growth varies with the corrinoid added. Cobalamin (vitamin B12) was preferred by all three enzymes, and the extent to which other corrinoids can be used was variable. S. meliloti can thus be used for future studies of corrinoid specificity by individually replacing the genes encoding each of the corrinoid-dependent enzymes examined here with those of different bacteria, and repeating the growth assays. We purified recombinantly expressed S. meliloti corrinoid-dependent methylmalonyl-CoA mutase (MCM) enzyme and reconstituted its activity in vitro. We conducted fluorescence-based corrinoid binding assays with three purified corrinoids and found variation in binding affinities of the enzyme for the five corrinoids tested. Objective 2: We used a fluorescence-based binding assay to measure the binding affinity of three different corrinoids for a single riboswitch RNA. The results show differences in binding of the RNA to different corrinoids. We explored other methods of measuring riboswitch-corrinoid binding and riboswitch function in order to identify a method most suited for high-throughput analysis of multiple riboswitch RNAs with multiple corrinoids. Objective 3: In an attempt to establish a stable synthetic co-culture between a corrinoid-producing and corrinoid-requiring bacterium, we combined two S. meliloti mutant strains with mutually co-dependent corrinoid metabolisms. Strain "A" carried a mutation in cobN and thus was unable to synthesize the corrin ring, and contained a plasmid that overexpressed bluB, resulting in an overproduction of the lower ligand 5,6-dimethylbenzimidazole (DMB). Strain "B" carried a mutation in bluB. Thus, strain A was dependent on strain B for corrin ring production, while strain B required strain A for DMB synthesis. The two strains were co-cultured under conditions in which neither strain was viable when cultured alone. Repeated passaging of these strains revealed that strain B began to dominate the co-culture after a single passage, and the abundance of strain A decreased upon each passage until becoming undetectable, likely due to excess levels of DMB produced by strain A. These results indicate that a delicate balance of metabolites is required to establish mutual co-dependence based on metabolites that are required in small amounts. Objective 4: We tested the hypothesis that guided biosynthesis (the incorporation of an exogenously added alternative lower ligand to form an alternative corrinoid) could be used to shift the corrinoid composition of a microbial community. We introduced three potential lower ligands: benzimidazole (Bza), dimethylbenzimidazole (DMB), and p-cresol (Cre) into the gut of the dampwood termite Zootermopsis and analyzed the corrinoid composition by liquid chromatography-tandem mass spectrometry. When Bza was added, we found that the levels of some of the corrinoids were altered, with a 6-fold increase in [Bza]Cba and a decrease in several other corrinoids, including a nearly complete elimination of [Ade]Cba. DMB and Cre had more modest effects on the corrinoid composition. Thus, we have shown that the corrinoid composition of this community can be altered by the addition of a lower ligand base. Guided biosynthesis could therefore be used to test the effect of corrinoid manipulation on community composition.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Mehta AP, Abdelwahed SH, Fenwick MK, Hazra AB, Taga ME, Zhang Y, Ealick SE, Begley TP. 2015. Anaerobic 5-Hydroxybenzimidazole Formation from Aminoimidazole Ribotide: An Unanticipated Intersection of Thiamin and Vitamin B?? Biosynthesis. J Am Chem Soc. 2015 Aug 26;137(33):10444-7.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Hazra AB, Han AW, Mehta AP, Mok KC, Osadchiy V, Begley TP, Taga ME. 2015. Anaerobic biosynthesis of the lower ligand of vitamin B12. Proc Natl Acad Sci U S A. 2015 Aug 25;112(34):10792-7.
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