Patent Application
20220364135
The present disclosure is directed to modified Propionibacterium and methods of use, including use to produce vitamin B12. Methods of increasing vitamin B12 production in a bacterium comprising a vitamin B12 riboswitch are also contemplated.
Incorporated by reference in its entirety is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: ASCII (text) file named 54547_Seqlisting.txt; Size: 26,555 bytes, created on Aug. 27, 2020.
Propionibacteria are gram-positive, facultative anaerobic, non-motile microorganisms with high GC contents, which can be taxonomically classified into the cutaneous (e.g., Propionibacterium acnes) and dairy bacterial species (e.g., Propionibacterium freudenreichh) (20, 21). P. freudenreichii, along with Pseudomonas species, is the major producer of vitamin B12 (VB12) and thus, has been widely used for industrial fermentation (2, 17).
Vitamin B12 (VB12), also known as cobalamin, is uniquely synthesized by some bacteria and archaebacteria, and is a crucial cofactor for critical enzymes catalyzing numerous transmethylation and the biochemical reactions(1, 2). VB12 structurally consists of a corrin ring, in which the cobalt is positioned centrally and coordinated with upper and lower ligand made-up of 5,6-dimethylbenzimidazole (DMB) (1, 3, 4). Both in bacterial and mammalian cells, methionine synthase for biosynthesis of S-adenosylmethionine (SAM), and methylmalonyl-CoA mutase converting methylmalonyl-CoA to succinyl-CoA, are highly dependent on VB12 for their metabolic activities (5). This renders VB12 to be essentially required for the biosynthesis of nucleic acids and the amino and fatty acid metabolisms. Henceforth, VB12 deficiency is critically associated with micronuclei formation and chromosomal abnormalities (6-8) and highly contributes to adverse pregnancy along with the neurological morbidity and death of neonates (9-14).
In one aspect, described herein is a Propionibacterium that has been modified to overproduce vitamin B12. In some embodiments, the Propionibacterium is P. freudenreichii. In some embodiments, the Propionibacterium is P. UF1. In some embodiments, the Propionibacterium comprises a mutation in a vitamin B12 riboswitch of the bacterium. The mutation decreases the activity of the vitamin B12 riboswitch. In some embodiments, the vitamin B12 riboswitch is cbiMCbl. In some embodiments, the mutation results in a deletion of stem loop 1 (SL1) of the cbiMCbl riboswitch. In some embodiments, the vitamin B12 riboswitch retains sequences encoding stem loops 2 and 3 of the vitamin B12 riboswitch.
Compositions comprising a modified Propionibacterium that has been modified to overproduce vitamin B12 are also contemplated. In some embodiments, the composition is a food product. In some embodiments, the composition is a beverage.
In another aspect, described herein is a method of increasing vitamin B12 production in a bacterium, the method comprising: (a) providing a bacterium comprising a vitamin B12 riboswitch; and (b) introducing a mutation in a vitamin B12 riboswitch that decreases activity of the vitamin B12 riboswitch, thereby increasing vitamin B12 production. In some embodiments, the method further comprises culturing the bacterium under conditions sufficient to produce vitamin B12. In some embodiments, the method further comprises isolating the vitamin B12 produced from the bacterium. In some embodiments, the bacterium is Pseudomonas dentrificans, Rhodobacter capusulatus, Rhodobacter sphaeroides, Sinorhizobium meliloti, Salmonella typhimurium, Bacillus megaterium, Propionibacterium shermanii, Thermotoga sp. RQ2, Thermotoga maritima MSB8, Thermotoga neapolitana, Thermotoga petrophila, Escherichia coli, Thermotoga naphthophila, Thermotoga thermarum, Thermotoga lettingae, Fervidobacterium nodosum, Thermosipho melanesiensis, Thermosipho africanus, Kosmotoga olearia, Mesotoga prima, Petrotoga mobilis. In some embodiments, the bacterium is Propionibacterium shermanii or Sinorhizobium meliloti. In some embodiments, the bacterium is Propionibacterium. In some embodiments, the bacterium is P. freudenreichii. In some embodiments, the bacterium is P. UF1. In some embodiments, the vitamin B12 riboswitch is cbiMCbl. In some embodiments, the mutation is deletion of stem loop 1 (SL1) of the cbiMCbl riboswitch. In some embodiments, the bacterium does not naturally produce vitamin B12 but has been genetically engineered to produce vitamin B12 (e.g., Escherichia coli and Pseudomonas dentrificans).
In another aspect, described herein is a method of producing vitamin B12, the method comprising (a) culturing a modified Propionibacterium described herein under conditions sufficient to produce vitamin B12; and (b) isolating vitamin B12 from the bacterium. In some embodiments, the bacterium is P. freudenreichii. In some embodiments, the bacterium is P. UF1.
In another aspect, described herein is a method of increasing vitamin B12 production in the gut of a mammalian subject comprising administering to the subject a composition described herein.
In yet another aspect, described herein is a method of increasing vitamin B12 production in the gut of a mammalian subject comprising administering to the subject a composition comprising a Propionibacterium and an agent that inhibits the activity of a vitamin B12 riboswitch in the Propionibacterium. In some embodiments, the vitamin B12 riboswitch is cbiMCbl.
In the following detailed description, embodiments are described in sufficient detail to enable those skilled in the art to practice them, and it is to be understood that other embodiments may be utilized and that changes may be made without departing from the spirit and scope of the present subject matter. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of embodiments is defined only by the appended claims. Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, and the like). Furthermore, disclosure of a range includes disclosure of all subranges included within the broader range (e.g., 1 to 5 discloses 1 to 4, 1.5 to 4.5, 4 to 5, and the like).
Gut bacteria-associated metabolites maintain host immune and developmental homeostasis. As shown in the Examples herein, the cbiMCbl riboswitch regulates the transcriptional and translational machineries of the cobA operon, controlling bacterial vitamin B12 (VB12) biosynthesis. As demonstrated herein, molecular modification of this riboswitch significantly enhanced VB12 production within the commensal P. UF1.
In one aspect, described herein is a bacterium that has been modified to overproduce vitamin B12. The term “modified bacterium” refers to a bacterium altered, modified or engineered (e.g., genetically engineered) such that it exhibits an altered, modified or different genotype or phenotype (e.g., when the genetic modification affects coding nucleic acid sequences of the bacterium) as compared to the naturally-occurring or parent bacterium from which it was derived.
In some embodiments, the bacterium is P. freudenreichii. In some embodiments, the bacterium is modified to include a mutation in a vitamin B12 riboswitch, wherein the mutation decreases activity of the vitamin B12 riboswitch. As described in the Examples, introducing a mutation in the vitamin B12 riboswitch decreases activity of the vitamin B12 riboswitch, which results in overproduction of vitamin B12. Mutating the region of the bacterial genome that encodes the vitamin B12 riboswitch can include, for example, deletion, substitution or insertion of nucleic acids within the genome of the bacterium encoding the riboswitch to generate a mutant riboswitch with decreased activity.
In some embodiments, the mutation is deletion of a stem loop of the riboswitch. In some embodiments, the mutation is deletion of stem loop1 1 (SL1) of the riboswitch. In some embodiments the vitamin B12 riboswitch is cbiMCbl.
The term “overproduction” as used herein refers to the ability of a bacterium to produce a product (e.g., a metabolite such as vitamin B12) at a higher level than is normally produced in the bacterium, thereby resulting in a greater amount of product than normally found in the bacterium. In this regard, overproduction is achieved by, for instance and without limitation, deleting stem loop 1 (SL1) in the vitamin B12 riboswitch, thereby increasing vitamin B12 production. An exemplary method of introducing a mutation in a vitamin B12 riboswitch is described in the Examples.
It will be appreciated that other bacterium comprise vitamin B12 riboswitches, and that the methods described herein are applicable to other bacterium. For example, in some embodiments, the bacterium is In some embodiments, the bacterium is Pseudomonas dentrificans, Rhodobacter capusulatus, Rhodobacter sphaeroides, Sinorhizobium meliloti, Salmonella typhimurium, Bacillus megaterium, Propionibacterium shermanii, Thermotoga sp. RQ2, Thermotoga maritima MSB8, Thermotoga neapolitana, Thermotoga petrophila, Escherichia coli, Thermotoga naphthophila, Thermotoga thermarum, Thermotoga lettingae, Fervidobacterium nodosum, Thermosipho melanesiensis, Thermosipho africanus, Kosmotoga olearia, Mesotoga prima, Petrotoga mobilis. In some embodiments, the bacterium is Propionibacterium shermanii or Sinorhizobium meliloti. In some embodiments, the bacterium is Propionibacterium. In some embodiments, the bacterium is P. freudenreichii. In some embodiments, the bacterium is P. UF1. In some embodiments, the vitamin B12 riboswitch is cbiMCbl. In some embodiments, the mutation is deletion of stem loop 1 (SL1) of the cbiMCbl riboswitch. In some embodiments, the bacterium does not naturally produce vitamin B12 but has been genetically engineered to produce vitamin B12 (e.g., Escherichia coli and Pseudomonas dentrificans). See Fang et al., Microb. Cell Fact, 16:15, 2017; Fang et al., Nat. Commun. 9:4917, 2018 and Xia et al., Bioprocess. Biosyst. Eng. 38:1065-73, 2015, the disclosures of which are incorporated herein by reference in their entireties.
A riboswitch is a region in an mRNA molecule that can directly bind a small target molecule, the binding of which modulates production of the encoded gene product. The small target molecules include, among others, vitamins, amino acids and nucleotides, and the binding is selective through a conserved sensor domain. Upon binding, the conformation of a variable “expression platform” coupled to the sensor domain is changed, and this can affect different modes of gene expression control including transcription termination, translation initiation or mRNA processing. Notably, riboswitches exert their functions without the need for protein cofactors. In most cases, they act in feedback regulation mechanisms: once the level of an end product (e.g., vitamin B12) in a metabolic pathway rises, riboswitch binding occurs, triggering a repression of gene expression in the same pathway. The substrate specificity of riboswitches is extremely high, allowing them to perform their activity amid the presence of numerous related compounds.
Another aspect of the disclosure is directed to a method of increasing vitamin B12 in a bacterium. This method comprises introducing a mutation in the vitamin B12 riboswitch that decreases activity of the vitamin B12 riboswitch and culturing the bacterium under conditions sufficient to produce vitamin B12.
A variety of culture systems are known in the art, including T-flasks, spinner and shaker flasks, roller bottles and stirred-tank bioreactors. Roller bottle cultivation is generally carried out by seeding cells into roller bottles that are partially filled (e.g., to 10-30% of capacity) with medium and slowly rotated, allowing cells to attach to the sides of the bottles and grow to confluency. The cell medium is harvested by decanting the supernatant, which is replaced with fresh medium. Anchorage-dependent cells can also be cultivated on microcarrier, e.g., polymeric spheres, that are maintained in suspension in stirred-tank bioreactors. Alternatively, cells can be grown in single-cell suspension.
Culture medium may be added in a batch process, e.g., where culture medium is added once to the cells in a single batch, or in a fed batch process in which small batches of culture medium are periodically added. Medium can be harvested at the end of culture or several times during culture. Continuously perfused production processes are also known in the art, and involve continuous feeding of fresh medium into the culture, while the same volume is continuously withdrawn from the reactor. Perfused cultures generally achieve higher cell densities than batch cultures and can be maintained for weeks or months with repeated harvests.
Host cells may be cultured using standard media well known to the skilled artisan. The media will usually contain all nutrients necessary for the growth and survival of the cells. Typically, an antibiotic or other compound useful for selective growth of transformed cells is added as a supplement to the media. The compound to be used will often be dictated by the selectable marker element present on the plasmid with which the host cell was transformed. For example, where the selectable marker element is kanamycin resistance, the compound added to the culture medium will be kanamycin. Other compounds for selective growth include ampicillin, tetracycline, geneticin, and neomycin.
The amount of vitamin B12 produced by a host cell can be evaluated using standard methods known in the art. Such methods include, without limitation, enzyme-linked immunosorbent assays (ELISAs), capillary electrophoresis (CE), radioisotope and mass spectrometry High Performance Liquid Chromatography (HPLC) separation, chemiluminescence (CL), absorption, fluorescence, surface plasmon resonance (SPR) and Raman spectroscopy.
Vitamin B12 produced by the bacterial cell can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y. (1982), Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc. N.Y. (1990)).
In another aspect, the increased production of vitamin B12 occurs in vivo (e.g., in the gut of a mammalian subject). In that regard, in one embodiment, the method comprises administering or ingesting a modified bacterium having a mutated vitamin B12 riboswitch, such as the modified bacteria described herein. Alternatively, the method comprises administering (or ingesting) an unmodified bacterium that produces vitamin B12 (e.g., a Propionibacterium such as P. UF1) in combination with an agent that inhibits the activity of a vitamin B12 riboswitch in the bacterium. Exemplary agents include small molecules and inhibitory RNA (RNAi) In some embodiments, the bacterium and the agent are administered (or ingested) at the same time. In some embodiments, the bacterium and the agent are administered sequentially (e.g., at separate times). In some embodiments, the bacterium and the agent are administered within minutes (e.g., about 1 minutes, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes) or hours (e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours, about 36 hours or about 48 hours) of each other.
Compositions comprising the modified bacterium described herein are also contemplated. In various aspects, the composition comprises live cultures of the bacteria, freeze-dried bacteria, or killed bacteria comprising the vitamin B12. The disclosure herein relating to compositions of modified bacteria also apply to compositions of unmodified bacteria, such as the unmodified bacteria described above.
In some embodiments, the composition can be in the form of capsules, pills, tablets, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an elixir or syrup, etc. In some embodiments, the composition is a food product, such as a probiotic food product. A “probiotic food product” refers to a food product which contains microorganisms associated with beneficial effects to humans and animals upon ingestion. Optionally, the food product is a beverage. The food or drink product may be a children's nutritional product such as a follow-on formula, beverage, milk, yogurt, fruit juice, fruit-based drink, chewable tablet, cookie, cracker, or a milk powder or the product may be an infant's nutritional product, such as an infant formula.
Compositions described herein can be administered by any means to deliver the composition to the intestine of a subject (e.g., oral administration, ingestion, or instillation directly into the intestines of the subject).
The modified bacterium (or composition comprising the modified bacterium) is administered to (or ingested by) a subject in need thereof in an effective amount which achieves a desired biological response in a clinically relevant time frame. In some embodiments, a bacterium that produces vitamin B12 (e.g., a Propionibacterium such as P. UF1) or composition comprising the bacterium is administered to (or ingested by) a subject in need thereof in combination with an agent that inhibits the activity of a vitamin B12 riboswitch in the bacterium.
An “effective amount” refers to the amount of the bacterium (whether modified or unmodified in combination with an agent that inhibits the activity of the vitamin B12 riboswitch in the bacterium), for example, P. freudenreichii, which is effective in producing beneficial effects in a subject ingesting the bacterium compared to a subject not exposed to the bacterium. In some embodiments, the beneficial effect is host intestinal homeostasis.
An effective amount of a modified bacterium described herein (or unmodified bacterium in combination with an agent that inhibits the activity of the vitamin B12 riboswitch in the bacterium) is determined based on the intended goal and may vary with the subject to which the composition is administered. The term “unit dose” refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the beneficial effect in association with its administration, for example, the appropriate route and treatment regimen. Generally, the dosage of the modified bacterium (or unmodified bacterium in combination with an agent that inhibits the activity of the vitamin B12 riboswitch in the bacterium) will vary depending upon such factors as the subject's age, weight, height, sex, general medical condition and medical history.
The effective amount of a bacterium can be expressed as an absolute number, for example, colony forming units (CFU), or as a body weight based dosage, for example CFU/Kg of body weight of the subject. Typically, an effective amount of the bacterium is about 104 to about 1012 CFU, about 105 to about 1011 CFU, about 106 to about 1010 CFU, about 108 to about 1010 CFU or about 108 to about 1012 CFU. In a specific embodiment, the effective amount is about 104 to about 1012 CFU/Kg, about 105 to about 1011 CFU/Kg, about 106 to about 1010 CFU/Kg, about 108 to about 1010 CFU/Kg or about 108 to about 1012 CFU/Kg of the body weight of the subject to which the composition is administered. In one embodiment, the effective amount is about 1012 to about 1013 CFU/Kg of the body weight of the subject to which the composition is administered.
In some embodiments, an effective amount comprises administration of multiple doses of a bacterium. The pharmaceutically effective amount may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or more doses of a composition comprising a bacterium. In some embodiments, doses are administered over the course of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, 30 days, or more than 30 days. Moreover, treatment of a subject with a therapeutically effective amount of a bacterium can include a single treatment or can include a series of treatments. It will also be appreciated that the effective dosage of a bacterium used for treatment may increase or decrease over the course of a particular regimen.
Materials and Methods:
Bacterial strains and growth: Escherichia coli NEB 5-alpha (New England BioLabs, MA) used for plasmid construction and E. coli Rosetta (DE3) (Sigma-Aldrich, St. Louis, Mo.) used for protein expression were grown in Luria-Bertani (LB) medium at 37° C. The P. UF1 and its genetically modified strains were grown at 30° C. in MRS medium (Difco Laboratories, Detroit, Mich.) with 1% (w/v) sodium lactate (Thermo Fisher Scientific, Waltham, Mass.) or Poznan medium (46) in an anaerobic chamber (Model AS-580, Anaerobe Systems, Morgan, Hill, Calif.). When appropriate, antibiotics were added to the medium at the following final concentrations: 5 μg/ml chloramphenicol and/or 1 mg/ml hygromycin B for P. UF1 isogenic strains; 100 μg/ml ampicillin for E. coli isogenic strains.
For deleting cobA gene from P. UF1, a 644-bp internal fragment of cobA was PCR amplified from P. UF1 chromosome using primers cobA bam-F and cobA xba-R. The purified fragment was cloned into pUCC plasmid (25), generating suicide plasmid pUCC-cobA. Following electroporation into P. UF1 (25), the chloramphenicol resistant colonies were selected and the ΔcobA P. UF1 mutant was identified by PCR analysis using primers P1 and P2. For complementation of the cobA knockout bacterial strain, the cobA gene with the native promoter was integrated into ΔcobA P. UF1 chromosome at the amyE locus, and the resultant C-ΔcobA P. UF1 strain was identified by PCR analysis using primers P3 and P4.
To generate the cbiMCbl riboswitch-FbFP translational fusion constructs, the 5′ UTR (
For overexpressing cobA operon in ΔcobA P. UF1, the cobA operon with the 5′ UTR was PCR amplified from P. UF1 genomic DNA with primers cbimop-sbf-F and cbimop-hid-R. After digestion with SbfI and HindIII, the purified PCR products were cloned into pYMZ vector, and the resulting construct was electroporated into ΔcobA P. UF1 to obtain OW-operon strain. To investigate the effect of SL1 deletion on VB12 biosynthesis, the SL1 region was deleted from 5′ UTR and the resulting fragment, along with the cobA operon, was ligated by overlapping PCR using primers cbimop-sbf-F/ΔSL1-R and ΔSL1-Fcbimop-hid-R. The constructed plasmid was electroporated into ΔcobA P. UF1 to obtain OΔSL1-operon strain. All obtained strains are listed in Table 1.
Vitamin B12 extraction and analysis: Bacterial cultures were centrifuged at 15,000 g for 10 minutes at 4° C. and washed twice using PBS. The cells were disrupted by boiling for 15 minutes in 0.1 M phosphate buffer containing 0.01% potassium cyanide at pH 6.0. After centrifugation at 15,000 g for 2 min, the supernatants were collected and passed through 0.22 μm filters (Millipore, Burlington, Mass.). Vitamin B12 (VB12) in the filtrates was quantified by Agilent 1220 infinity II LC system comprised of an automated sampler (G4282B), gradient Pump (G4281 B) and VWD detector (G4284B). Separations were performed using the following mobile phase (46), 0.25 M NaH2PO4, pH 3.5 (phosphoric acid): methanol (75:25) in an Agilent C18 column (Eclipse Plus C18, 3.5 μm, 4.6×150 mm column) at 20° C. with a flow rate of 1.0 ml/min. The total HPLC run time for each sample was 15 minutes, and the injection volume was 20 μl. The detector wavelength was set at 362 nm. Quantitation was based on peak area and the standard curve of VB12. Data acquisition and analysis were done using Agilent ChemStation.
RNA Sequencing: For bacterial RNA-seq, total RNA was isolated from P. UF1 and ΔcobA P. UF1 strains and the DNA libraries were constructed essentially as previously described (25). The obtained cDNA libraries were sequenced on an Illumina HiSeq instrument (Illumina, Inc., San Diego, Calif.) at the University of Florida ICBR NextGen DNA Sequencing Core Facility. Sequence analyses of bacterial samples were performed as described previously (25), with some modifications. Principal component analysis was performed upon regularized-log-transformation of the count data using Python Scikit-learn package. Pathway analysis was analyzed using an in-house informatics pipeline, which modified from Mummichog's pathway analysis module (47). Briefly, the Enzyme Commission number (EC number) of each annotated gene of P. UF1 genome was extracted from databases including KEGG and BioCyc. The genes were categorized into pathways and modules from KEGG database based on the EC number. 71 significantly upregulated and 73 downregulated genes in P. UF1 compared to ΔcobA P. UF1 were used as input for pathway analysis (p<0.05, fold change >2). Pathway enrichment p-values were calculated based on permutation in Mummichog's pathway analysis module. The pathways represented by at least two genes and enriched at p<0.05 are presented. Heatmap analysis was generated on selected significant genes (p<0.05, fold change >1.5) related to the indicated metabolic pathways.
High-resolution metabolomics analysis: Bacterial samples were processed and metabolites were extracted as previously described (25). Adaptive processing of Liquid Chromatography-Mass Spectrometry (apLCMS) was used to perform peak detection, noise filtering, mass to charge (m/z) ratio and retention time alignment and feature quantification. In-house informatics pipelines were used to quality control and clean up the feature table. PCA was performed on log 2-transformed intensity values of all metabolite features. Subsequently, student's t-test was used to evaluate difference of metabolite features between P. UF1 and ΔcobA P. UF1. False discovery rate (FDR) was calculated using the Benjamini-Hochberg method. FDR-adjusted p-values less than 0.05 were considered significant. Metabolic pathway analysis was performed by Mummichog (v1.0.5) (47), with a modified adduct function to include M+H—CN[2+], an experiment-verified metabolite adduct that matches VB12. 283 significant metabolite features were used as input to Mummichog (FDR adjusted p<0.05, student's t-test). The total list of features was used as reference. Pathway enrichment p-values were calculated in Mummichog based on permutation. The pathways represented by at least two metabolite features and enriched at p<0.05 are presented. KEGG database was used to identify metabolite features related to significant pathways identified by Mummichog. Identified metabolite features with adduct ions of M+H[1+] or M+H—CN[2+], were selected to show in the scatter plot, with each dot representing the log 2-transformed intensity values.
FbFP reporter assays: To perform FbFP fluorescence intensity measurement, bacteria were cultivated for three days in triplicate, washed, and resuspended with PBS. The excitation and emission wavelengths of FbFP were set at 452 nm and 495 nm on a microplate reader (BioTek, Winooski, Vt.), respectively (48). FbFP intensity at 495 nm was normalized by corresponding OD600 values for each sample. P. UF1 strain with an empty vector was used as the baseline for calculation of fluorescence intensity.
Preparation of anti-CobA serum antibodies: To generate CobA polyclonal antibodies, the pET21b-cobA expression plasmid was constructed by PCR amplification of the cobA gene using primers 21-cobA-bamF and 21-cobA-xhoR (provided in Table 2).
Following transformation into E. coli Rosetta(DE3), CobA expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma Aldrich, St. Louis, Mo.). Cell lysates were separated by 12% SDS-PAGE, and the CobA proteins were excised from the gel and used to immunize C57BL/6 mice, resulting in anti-CobA serum antibodies.
Western blot: Bacteria were cultivated for three days, and washed three times, followed by lysozyme digestion (10 mg/ml) for 2 hours at 37° C. Cell lysates were separated by 12% SDS-PAGE, transferred to PVDF membranes (Sigma Aldrich, St. Louis, Mo.), and blocked with Odyssey blocking buffer (Li-Cor Biosciences, Lincoln, Nebr.). Subsequently, membranes were incubated with anti-HIS-tag antibody (Thermo Fisher Scientific, Waltham, Mass.), anti-TetR antibody (Takara Bio, Mountain view, CA), anti-CobA serum antibodies, or anti-LspA serum antibodies (Stock in our lab) for 2 h at room temperature in the blocking buffer with 0.02% Tween 20. After washing with TBST (20 mM Tris, 150 mM NaCl and 0.1% Tween 20, pH 7.4), membranes were incubated with IRDye 680RD goat anti-mouse secondary antibody (Li-Cor Biosciences, Lincoln, Nebr.) for 1 hour at room temperature in the blocking buffer with 0.02% Tween 20. After washing with TBS, the proteins were detected using an Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln, Nebr.). LspA was used as an internal control.
Quantitative RT-PCR: Total RNA was extracted from various bacterial cultures using Trizol reagent (Thermo Fisher Scientific, Waltham, Mass.), and the RNA mixtures were further purified using the Aurum Total RNA Mini Kit (Bio-Rad, Hercules, Calif.). The cDNA was synthesized from total RNA using iScript Advanced cDNA Synthesis Kit (Bio-Rad, Hercules, Calif.). Quantitative real-time was accomplished with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, Calif.) on a CFX96 real-time PCR system (Bio-Rad, Hercules, Calif.) using primers listed in Table 2 above. Results were normalized to those obtained from groL2 gene.
Biochemical studies demonstrated that CobA is essential for the conversion of uroporphyrinogen III to precorrin-2, which is the branch point between the biosynthesis of vitamin B12 (VB12) and siroheme (18) (
The cobA gene was deleted from the bacterial chromosome by homologous recombination with a single crossover event, resulting in ΔcobA P. UF1 (
Vitamin B12 (VB12), as an enzymatic cofactor, critically regulates the transcriptomic and metabolic machineries in diverse bacteria (29), including human gut microbiota. To underscore the central implication of CobA in regulating gene expression within P. UF1, RNA sequencing (RNA-Seq) was performed (25).
Overall, principal component analysis (PCA) plot exhibited a distinct separation of the global transcriptomes of P. UF1 and ΔcobA P. UF1 (
Further, to investigate whether the observed changes in bacterial transcriptome correlate with metabolomic modifications, high-resolution liquid chromatography-mass spectrometry (LC-MS) was employed to analyze the metabolic profiling of ΔcobA P. UF1 and P. UF1. Here, PCA demonstrated a distinct clustering of the two metabolomes at day 3 and 6 of bacterial cultures (
In bacteria such as Escherichia coli (E. col′) and Salmonella typhimurium (S. typhimurium), VB12 interacts with the 5′ UTR of VB12 biosynthesis operon to repress the translation of the corresponding genes, including cob and btuB operon (30, 31). Demonstrating the central role of cobA in controlling VB12 biosynthesis within P. UF1 prompted us to determine the feedback regulation of cobA operon by VB12 (
The cobA operon harbors cbiMNQOA genes encoding proteins critical for cobalt transport and precorrin-2 biosynthesis (
VB12 possesses analogues such as cyanocobalamin (manufactured form), methylcobalamin (active form), hydroxocobalamin (storage form) and adenosylcobalamin (active form) (14). To elucidate whether there would be a differential regulation by aforementioned VB12 analogues, CbiM-ΔcobA and FbFP-ΔcobA bacterial cultures (Table 1) were treated with various concentrations of the VB12 analogues to analyze the cbiM and FbFP expression. As shown in
Riboswitches are noncoding RNA (ncRNA) regulatory elements that specifically bind small-molecular ligands such as VB12 to modulate gene expression (24). To identify potential regulatory element(s), the 5′ UTR (309 bp) of cobA operon was used to perform the comparative analysis. Conserved-secondary structure and sequence homology analyses demonstrated the presence of a potential VB12 riboswitch (Rfam accession, RF00174, 140 bp), designated as cbiMCbl riboswitch, which contains three major stem-loops (SL), including SL1, SL2, and SL3 (
VB12-element exhibits a conserved RNA regulatory sequence in many VB12 riboswitches of numerous microorganisms (15, 29). To investigate whether VB12-element exists within cbiMCbl riboswitch, the known VB12 riboswitches and cbiMCbl riboswitch were compared by sequence alignment analysis using LocARNA, whereby a conserved VB12-element and various secondary structures (e.g., Pkn) were identified within cbiMCbl riboswitch (
Thus far, it is unclear if the regulatory activity of cbiMCbl riboswitch would be dependent on Watson-Crick base pairing between the regulatory domains (Pkn and antisequester) and their complementary domains (Pkn′ and RBS-sequester). Thus, to shed light on this notion, we introduced a second mutation into these single mutants to rescue their base pairing (
Having determined that VB12 riboswitch controls gene expression through transcriptional and/or translational modifications (24, 34), the impact of the cbiMCbl riboswitch on the transcription of cobA operon was further assessed by qRT-PCR. Obtained data revealed that the mRNA levels of most of the genes, including cbiM, cbiN, cbiO and cbiQ, were significantly repressed in a VB12-dependent manner within P. UF1 (
Discussion
Mechanisms involved in bacterial molecular machinery are critically directing the metabolic circuits that regulate the bacterial homeostasis and their stable abundancy, all of which significantly contribute to human health (3, 35-37). One of these metabolites that is biosynthesized by only a few gut bacteria is VB12, which crucially impacts the cross talk between gut microbes and the host (3, 38). Although previous data demonstrated how pathogens, including Salmonella (30, 39), regulate the biosynthesis of this vitamin, the control of VB12 biosynthesis in bacteria with bifidogenic properties, particularly probiotic bacteria, is currently obscure. Thus, to shed light on the mechanistic complexes regulating VB12 in the newly discovered P. UF1 bacterium that controls the local and peripheral immune homeostasis in steady state and during gut infection, we first focused on the genomic region of bacterial CobA. CobA catalyzes the S-adenosyl-L-methionine (SAM)-dependent bismethylation of uroporphyrinogen III synthase (40) to form precorrin-2, the primary precursor of VB12 (18). Thus, deleting cobA from the bacterial chromosome fully abolished VB12 production in ΔcobA P. UF1 strain. Furthermore, VB12 was restored by complementing ΔcobA P. UF1 with cobA gene, illuminating the critical role of cobA in bacterial VB12 synthesis. Further, to elucidate the significance of cobA in VB12 production and its involvement in the regulation of bacterial metabolomic pathways, particularly propionate synthesis, RNA-seq analysis (25) demonstrated cobA deficiency indeed critically altered the transcriptome of P. UF1 whereby metabolic pathways, including porphyrin, chlorophyll, propionate, cysteine, methionine and sulfur metabolisms, were decisively impacted in this bacterium. This was also confirmed through metabolomic analysis of P. UF1 and ΔcobA P. UF1, whereupon various cellular metabolisms, particularly propionate, were impaired, once again highlighting the relevance of cobA in controlling VB12 molecular events that in turn influence the bacterial metabolic homeostasis.
Further, the Examples provided herein also demonstrate that endogenous and exogenous VB12 tightly controlled the expression of cobA operon via its 5′ UTR within P. UF1 bacterium. As shown herein, VB12 completely inhibited the expression of downstream genes by a novel cbiMCbl riboswitch at 750 μM, which was significantly higher than env8HyCbl in E. coli (34).
VB12 analogues displayed the similar regulatory activities in the riboswitch of P. UF1 but not in E. coli (33), possibly as a result of diverse sequences and secondary structures of the VB12 riboswitch. Here, the structure-based analyses revealed that SL1 and SL3 were highly required for the regulatory function of cbiMCbl riboswitch, which belong to receptor domains conserved within the VB12 riboswitches from E. coli and S. typhimurium (29, 33, 41). Further, we clearly demonstrated that Pkn in SL2 and the complementary Pkn′ were crucial regulatory domains within the cbiMCbl riboswitch, whose regulatory function was notably not dependent on Watson-Crick base pairing between the two domains. In contrast, the regulation of env8HyCbl highly depends on the Watson-Crick base pairing of “kissing loop” between SL2 and RBS region (33, 34), indicating cbiMCbl riboswitch within P. UF1 may employ a new molecular mechanism to control gene expression. Interestingly, a novel Watson-Crick base pairing between the RBS-sequester and antisequester was identified in the Examples herein, whose base paring was essentially required for regulatory activities of the riboswitch. In E. coli, it has been well documented that the env8HyCbl riboswitch regulates gene expression at transcriptional and translational levels (33, 34), as found for cbiMCbl riboswitch. In Listeria monocytogenes and S. typhimurium, VB12 riboswitches don't control the transcription of the downstream genes (24, 41), indicating that various VB12 riboswitches may display a distinct regulatory model for gene expression in bacteria with different natures and functions, mainly pathogenic, or beneficial bacteria such probiotics.
The probiotics are defined as live microbial feed supplements with beneficial properties, which potentially benefit the host when administered in adequate amounts (42). Propionibacterium species are currently of great interest for their beneficial effects as probiotics and are applied to human dietary consumption, including Swiss cheese (43). Here, we demonstrated that cbiMCbl riboswitch controlled the expression of the cobA operon at transcriptional and translational levels within this bacterium and that the genetic modification of this riboswitch significantly enhances VB12 biosynthesis within P. UF1. Henceforth, it is reasonable to speculate that this bacterium abundantly synthesizing VB12 may critically contribute to the observed immune homeostasis in the host.
VB12 can be used by more than 80% of gut microbiota (3), suggesting that this vitamin can be utilized to support the healthy ecology of the gut microbiota involved in the intestinal immune homeostasis of the host (3, 44). Here, we clearly demonstrated how precisely VB12 biosynthesis was regulated within cobA through a new riboswitch, cbiMCbl, within P. UF1. This riboswitch may serve as a novel target to increase the levels of VB12 by ingesting a probiotic bacterium with bifidogenic properties that can contribute to enhanced cross-feeding of neighboring gut bacteria, that potentially mitigate the symptoms of intestinal disorders in the near future (45).
The present application claims the benefit of priority to U.S. Provisional Application No. 62/894,301, filed Aug. 30, 2019, the disclosure of which is incorporated herein by reference in its entirety.
This invention was made with government support under DK109560 awarded by The National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US20/48324 | 8/28/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62894301 | Aug 2019 | US |
