High-throughput detection and quantification of vitamin B12in microbiome isolates using Escherichia coli

High-throughput detection and quantification of vitamin B12 in microbiome isolates using Escherichia coli | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results High-throughput detection and quantification of vitamin B 12 in microbiome isolates using Escherichia coli Katarzyna Hencel , Matthew Sullivan , View ORCID Profile Alper Akay doi: https://doi.org/10.1101/2025.04.08.647760 Katarzyna Hencel 1 School of Biological Sciences, University of East Anglia , Norwich, NR4 7TJ, UK 2 Centre for Microbial Interactions , Norwich Research Park, Norwich, NR4 7UJ Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: k.hencel{at}uea.ac.uk matthew.sullivan{at}uea.ac.uk a.akay{at}uea.ac.uk Matthew Sullivan 1 School of Biological Sciences, University of East Anglia , Norwich, NR4 7TJ, UK 2 Centre for Microbial Interactions , Norwich Research Park, Norwich, NR4 7UJ Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: k.hencel{at}uea.ac.uk matthew.sullivan{at}uea.ac.uk a.akay{at}uea.ac.uk Alper Akay 1 School of Biological Sciences, University of East Anglia , Norwich, NR4 7TJ, UK 2 Centre for Microbial Interactions , Norwich Research Park, Norwich, NR4 7UJ Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Alper Akay For correspondence: k.hencel{at}uea.ac.uk matthew.sullivan{at}uea.ac.uk a.akay{at}uea.ac.uk Abstract Full Text Info/History Metrics Preview PDF Abstract Vitamin B 12 is an essential micronutrient produced only by prokaryotes, and animals must acquire it from their diet. Vitamin B 12 is critical for the synthesis of methionine and propionyl-CoA metabolism. In humans, vitamin B 12 deficiency has been linked to many disorders, including infertility and developmental abnormalities. The growing trend towards plant-based diets and the ageing populations increase the risk of vitamin B 12 deficiency, and therefore, there is an increasing interest in understanding vitamin B 12 biology. Accurate approaches for detecting and quantifying vitamin B 12 are essential in studying its complex biology, from its biogenesis in Bacteria and Archaea to its effects in complex organisms. Here, we present an approach using the commonly available E. coli methionine auxotroph strain B834 (DE3) and a multi-well spectrophotometer to detect and quantify vitamin B 12 from biological samples at picomolar concentrations. We further show that our quantification method for vitamin B 12 is sufficient to reveal important differences in the production of vitamin B 12 from vitamin B 12 -synthesising bacteria commonly found in the microbiome of wild Caenorhabditis elegans isolates. Our results establish a high-throughput and simple assay platform for detecting and quantifying vitamin B 12 using the E. coli B834 (DE3) strain. Introduction Cobalamin (the natural form of vitamin B 12 ) is, structurally, the most complex vitamin. It is only produced by bacteria and archaea and requires more than a dozen enzymes for its biogenesis. In many organisms, cobalamin (vitamin B 12 hereafter) is essential for the function of two critical enzymes: methionine synthase, which regenerates methionine in the cells from homocysteine, and methylmalonyl-CoA mutase, which converts propionyl-CoA into succinyl-CoA. In humans, vitamin B 12 deficiency has been linked to multiple diseases, including anaemia, infertility, and developmental and neurological disorders ( 1 , 2 ). Although clinical levels of vitamin B 12 deficiency are rare ( 3 ), the global increase in plant-based diets and the ageing populations are linked to reduced vitamin B 12 uptake, which is considered a growing global health risk that necessitates further molecular and medical research in vitamin B 12 and its roles in human and animal physiology ( 4 , 5 ). Research on vitamin B 12 requires sensitive detection and quantification methods. Using vitamin B 12 auxotrophy in bacteria to quantify vitamin B 12 in biological samples has been a standard method since the 1940s when Lactobacillus leichamnnii was described by multiple groups as a suitable strain for vitamin B 12 quantification ( 6 – 8 ). The assay has been used to this day and is readily available through commercial routes. However, L. leichamnnii has complicated growing conditions, including its response to thymidine in the absence of vitamin B 12 ( 9 ). Another bacterial strain commonly used for vitamin B 12 assays is Salmonella typhimurium mutants, which lack the vitamin B 12 -independent methionine synthase, MetE ( 10 ). However, Salmonella strains grow under anaerobic conditions, often requiring additional apparatus. Another well-established assay uses the microalgae Euglena gracilis var. bacillaris ( 11 ). Although considered more accurate, this assay takes 4 to 7 days to complete and involves complicated growth conditions. Alternatively, methionine auxotroph strains of Escherichia coli (E. coli) can also be used for vitamin B 12 quantification ( 12 – 15 ). The advantages of utilising E. coli strains for vitamin B 12 assays include fast growth, which allows the assay to be performed overnight, and simple growth requirements, which do not involve specialised media preparation. However, there is limited information on the sensitivity and specificity of E. coli -based vitamin B 12 assays. Here, we present a high-throughput bacteria-based assay for vitamin B 12 detection and quantification using the methionine-auxotroph E. coli B834 (DE3) strain. This approach allows for simple and time-efficient detection and quantification of vitamin B 12 content in biological samples. We further utilise the method to quantify the vitamin B 12 content of different bacterial species commonly found with wild isolates of the nematode Caenorhabditis elegans (C. elegans) . Methods Bacterial strains, plasmids and culture media All bacteria and plasmids are listed in Table 2 and Table 3 , respectively. E. coli OP50, C. aquatica DA1877 and the isogenic Δ cbiA Δ cbiB mutant were grown in the soya-rich medium at 37 °C at 180 rpm agitation for the extract preparation. E. coli B834 (DE3) was grown in M9 minimal salts medium (KH 2 PO 4 , 15 g/L NaCl, 2.5 g/L Na 2 HPO 4 , 33.9 g/L NH 4 Cl, 5 g/L, 2 mM MgSO 4 , 0.1 mM CaCl 2 , 0.4 % glucose unless otherwise stated) supplemented with 400 nM L-methionine at 37 °C at 180 rpm. Strains from the CeMbio collection for the extract preparation were grown at 28 °C at 180 rpm in the vitamin B12-deficient, soya-rich medium (soya peptone 20 g/L, sodium chloride 5 g/L). View this table: View inline View popup Table 1. Bacterial strains used in the study View this table: View inline View popup Download powerpoint Table 2. Plasmids used in this study View this table: View inline View popup Table 3. Primers used in the study C. aquatica DA1877 vitamin B 12 -deficient mutant generation Oligonucleotide primers ( Table 3 ) with flanking 20 bp overhangs were designed to amplify upstream and downstream fragments from cbiA and cbiB of C. aquatica DA1877 using Benchling’s Gibson Assembly Wizard. The amplified fragments were introduced to the pFOK suicide vector through Gibson Assembly and transformed into E. coli JKE201. All constructs were verified by Sanger sequencing, followed by conjugation with C. aquatica DA1877 on LB supplemented with 100 µM diaminopimelic acid (DAP) to support E. coli JKE201 growth. Transconjugants were selected onto LB agar containing 100 µg/mL kanamycin. At least 3 transconjugants were grown in LB medium for 4 hours, followed by plating on no-salt LB agar plates (10 g/L tryptone, 5 g/L yeast extract, 15 g/L agar) supplemented with 20 % sucrose and 0.5 µg/mL anhydro-tetracycline. Candidate colonies were screened for deletions through PCR and verified by Sanger sequencing. Mutation in cbiB resulted in the out-of-frame deletion of 178 amino acids, while cbiA mutation resulted in complete gene removal. The Sanger sequencing trace files are available as Supplementary File 1. Bacterial lysate preparation Bacterial cultures for the E. coli B834 (DE3) assay were grown overnight and 1 OD unit (equivalent of 1 mL of culture with absorbance at 600nm of 1.0) was centrifuged at 15,000 rpm for 1 minute. The supernatant was removed, and the cells were resuspended in 50 µL of M9 minimal salts medium and boiled at 100 °C for 15 minutes, as previously described ( 22 ). After boiling, lysates were centrifuged at 15,000 rpm for 1 minute to remove debris, and the cooled supernatant was used as an extract for supplementation assays. E. coli B834 (DE3) Assay The assay was prepared in 96-well plates ( Greiner #655180) with the final volume of 200 µL of M9 minimal salts medium. Overnight cultures of E. coli B834 (DE3) grown in LB were back-diluted 1:100 into the wells and supplemented with either 2 µL of prepared bacterial lysates (extracts) or vitamin B 12 standard solutions used for the growth curves. The growth response was recorded over 20 hours at 37°C with 300 rpm agitation, with readings taken every 30 minutes using a SPECTROstar ® Nano plate reader (BMG Labtech) in matrix scan mode using a 2x2 scan matrix with 25 flashes per scan point and path length correction of 5.88mm for 200 µL volume. For blank corrections of optical density readings, control wells containing media without bacteria were included. Methylcobalamin (Thermo Scientific Chemicals, #A11176ME) was used for the vitamin B 12 standard curve. Visualisation and analysis Data was analysed and visualised using Prism 10 (Version 10.3.0). Results Analysis of E. coli B834 methionine and vitamin B 12 auxotrophy E. coli has two methionine synthase enzymes: the B 12 -dependent MetH and the B 12 -independent MetE. E. coli B834 (DE3) has a null mutation in the metE gene, making the bacteria solely dependent on either methionine or B 12 supplementation. To assess whether E. coli B834 (DE3) is suitable for vitamin B 12 detection and quantification, we tested the growth of this strain in response to various vitamin B 12 and methionine supplementations. We confirmed that E. coli B834 (DE3) can grow only when methionine or vitamin B 12 is present in the media ( Figure 1A ). We did not observe any significant difference in growth when B 12 was supplemented at concentrations ranging from 1 nM to 1000 nM, as determined by area under the curve analysis followed by one-way ANOVA with Holm-Sidak multiple comparison corrections ( Figure 1B ). Download figure Open in new tab Figure 1. The growth of E. coli B834 is dependent on methionine or vitamin B 12 and is titratable with vitamin B 12 concentration. E. coli B834 was cultured in M9 minimal media, without or with supplemental methionine or vitamin B 12 (1 nM – 1000 nM) as indicated (A) . The growth conditions were compared by Area Under the Curve analysis, followed by one-way ANOVA with Holm-Šidák multiple comparison corrections and P-values indicated (B) . E. coli B834 growth with vitamin B 12 supplementation at 1 nM and using 10-fold (C) and 2-fold serial dilutions (E) to test a broad range of sub-nanomolar vitamin B 12 concentrations. The growth of E. coli B834 in the 10-fold and 2-fold were compared by Area Under the Curve analysis followed by one-way ANOVA and Holm-Šidák multiple comparison corrections and comparisons are shown (D) and (F) , respectively. The data points are plotted as mean ± SEM from 3 biological replicates derived from 2 technical replicates. Using the growth of E. coli B834 as a measure of vitamin B 12 quantity We subsequently sought to test the utility of using the growth of E. coli B834 (DE3) as a highly sensitive biological method for detecting vitamin B 12 . To this end, we used M9 minimal media (devoid of methionine or vitamin B 12 ) and added vitamin B 12, supplemented at concentrations ranging from 0.00001 nM to 1 nM using 10-fold ( Figures 1C and 1D ) and 2-fold ( Figures 1E and 1F ) serial dilutions. Using this range, we determined that vitamin B 12 concentrations at and above 0.25 nM (250 pM) were sufficient to support the growth of the E. coli B834 (DE3) strain, as evidenced by a significant increase in area under the curve measurements throughout the growth period ( Figure 1F ). Next, we prepared a standard curve of vitamin B 12 concentrations between 0 and 0.4 nM to determine the limit of detection and quantification for vitamin B 12 using the E. coli B834 (DE3) strain. Compared to the unsupplemented media control, the limit of detection is 0.05 nM (50 pM) ( Figure 2A and 2B ), indicating that the growth of E. coli was detectable above background absorbance measurements. Increasing the carbon source from 0.4% glucose to 1.0% did not change the sensitivity of the growth assay ( Supplementary Figure 1A and 1B ). In summary, we have established that the growth of E. coli B834 (DE3) can be used to detect and quantify vitamin B 12 at picomolar concentrations. Download figure Open in new tab Figure 2. Use of E. coli B834 growth to detect and quantify vitamin B 12 production by C. aquatica . E. coli B834 was cultured in M9 minimal medium, without or with supplemental vitamin B 12 at concentrations between 0.025 nM – 0.4 nM ( A ) and compared using Area under the Curve analysis coupled with one-way ANOVA with Holm-Šidák multiple comparison corrections (B) . E. coli B834 culture was supplemented by bacterial cell-free extracts of C. aquatica DA1877, Δ cbiA Δ cbiB C. aquatica , and E. coli OP50 (C) and growth in the presence of extracts were compared by Area under the Curve analysis coupled with one-way ANOVA with Holm-Šidák multiple comparison corrections (D) . Area under the Curve data from vitamin B 12 standards in Panel B were used for Gompertz model fitting, further employed for vitamin B 12 quantification ( Y = YM*(Y0/YM)^(exp(-K*X) ), where YM is the maximum AUC score, Y0 is the minimum AUC score, K determines the lag time. The dashed green line corresponds to the 4.70 AUC score of the 10 -1 dilution of C. aquatica DA1877 extract, while the dashed blue line corresponds to the 5.20 AUC score of the 1:8 dilution of C. aquatica DA1877 extract, where 2 µL out of 50 µL of the 1 OD extract was used for quantification. In this model, YM = 8.606, Y0 = 0.7062, and K = 12.53. All data points are plotted with mean ± SEM from 3 biological replicates derived from 2 technical replicates. Using E. coli B834 (DE3) to quantify vitamin B 12 in biological samples C. elegans is a well-established model organism for studying the function of vitamin B 12 during animal development and for understanding the molecular pathways related to vitamin B 12 ( 24 – 28 ). C. elegans exclusively feeds on bacteria, and its uptake of vitamin B12 depends on the bacteria available in its environment as a food source. One such bacterium C. elegans feeds on in the wild is Comamonas aquatica (C. aquatica) DA1877, a known vitamin B 12 producer ( 28 ) that was isolated from soils ( 19 ). Mutations in the cbiA and cbiB genes, which code for cobyrinate a,c-diamide synthase and adenosylcobinamide-phosphate synthase enzymes, respectively, prevent C. aquatica from producing vitamin B 12 ( 28 ). As a negative control for our assay, we generated an isogenic Δ cbiA Δ cbiB mutant of DA1877 by deleting the cbiA and cbiB genes ( Supplementary Figure 2A-2C ). To confirm that our Δ cbiA Δ cbiB strain no longer produced vitamin B 12 , we utilised our E. coli B834 (DE3) approach to test for the presence of vitamin B 12 in cell-free extracts from C. aquatica . Briefly, bacterial cells were lysed by boiling, and cell-free extracts were added to E. coli B834 (DE3) in media devoid of vitamin B 12 or methionine. Using this approach, we assayed the vitamin B 12 levels of wild-type C. aquatica, C. aquatica Δ cbiA Δ cbiB and E. coli OP50, a different strain of E. coli commonly used as laboratory food for C. elegans but known to be a vitamin B 12 non-producer ( 28 , 29 ). As predicted, cell-free extracts of wild-type C. aquatica DA1877 supported vitamin B 12 -dependent growth of E. coli B834 (DE3), whereas the C. aquatica Δ cbiA Δ cbiB mutant and E. coli OP50 did not ( Figure 2C and 2D ). We further quantified the vitamin B 12 content from C. aquatica extracts by using 2-fold and 10-fold serial dilutions and comparing the relative growth of E. coli B834 (DE3) with C. aquatica extracts against growth with known concentrations of vitamin B 12 ( Supplementary Figure 2D and 2E ). Using the 2-fold and 10-fold dilutions combined with the Gompertz-modelled Area under the Curve analysis of the standard curve, we estimate the vitamin B 12 content of C. aquatica DA1877 to be approximately 25 nM per 1 OD unit (1 mL of culture at 1.0 OD 600nm ) of bacteria ( Figure 2E ). Our assays with vitamin B12, along with extracts from both vitamin B12-producing and non-producing bacteria, provided proof of concept for our method to detect vitamin B12 in complex biological samples by using the growth of E. coli B834 (DE3) as a proxy. Next, we applied this method to assess the vitamin B12 content of 12 bacterial strains from the CeMbio collection, all of which were isolated from C. elegans found in the wild ( 20 ). Four bacterial strains, Comamonas piscis BIGb0172, Pseudomonas berkeleyensis MSPm1, Pseudomonas lurida MYb11 and Ochrobactrum vermis MYb71, were predicted to produce vitamin B 12 based on their genomic sequences and predicted metabolic pathway analyses ( 20 , 29 ). Our analysis using the E. coli B834 (DE3) growth assay showed that C. piscis BIGb0172, P. berkeleyensis MSPm1, P. lurida MYb11, and O. vermis MYb71 are indeed vitamin B12 producers, because cell-free extracts from cultures of these bacteria were capable of supporting the growth of E. coli B834 (DE3) in a manner that relied on vitamin B12 ( Figure 3A and 3B ). Among these, extracts from C. piscis BIGb0172 and P. berkeleyensis MSPm1 supported the highest growth, while O. vermis MYb71 showed reduced growth, indicating that there may be variation in the amount of vitamin B 12 produced by these bacteria. In contrast, supplementing E. coli B834 (DE3) with extracts from the other eight CeMbio strains led to a complete absence of bacterial growth ( Figure 3A and 3B ). Download figure Open in new tab Figure 3. Quantification of vitamin B 12 levels in bacterial isolates from wild C. elegans E. coli B834 was cultured in M9 minimal media and supplemented with cell-free bacterial extracts of the indicated strains (A) . Growth was compared using Area Under the Curve analysis coupled with one-way ANOVA with Holm-Šidák multiple comparison corrections (B) . The data points are plotted with mean ± SEM from 3 biological replicates derived from 2 technical replicates corrected for blank readings. Bacterial isolates are as follows: Sphingobacterium multivorum BIGb0170, Comamonas piscis BIGb0172, Pantoea nemavictus BIGb0393, Enterobacter hormaechei CEent1, Sphingomonas molluscorum JUb134, Stenotrophomonas indicatrix JUb19, Chryseobacterium scophthalmum JUb44, Lelliottia amnigena JUb66, Pseudomonas berkeleyensis MSPm1, Acinetobacter guillouiae MYb10 Pseudomonas lurida MYb11, Ochrobactrum vermis MYb71. In summary, we show that our application of E. coli B834 (DE3) growth in minimal media can be used for rapid and high-throughput detection and relative quantification of vitamin B 12 in biological samples. Discussion Vitamin B 12 -dependent microorganisms are commonly used to detect and quantify vitamin B 12 in various formats ( 6 – 8 ). Using E. coli metE mutants for this purpose offers numerous advantages, including their commercial availability, rapid growth, and simple growth requirements. However, there is limited information on the sensitivity and reproducibility of E. coli metE -based vitamin B 12 assays. Here, we present a vitamin B 12 quantification assay using a readily available commercial strain of E. coli B834 (DE3) and widely used and inexpensive minimal media. The assay was developed in liquid culture using a 96-well plate format and a multi-well plate reader, allowing for reproducible analysis of many biological samples with a sensitivity as low as 50 picomolar concentration. The dependency on the growth of E. coli B834 (DE3) due to the presence of vitamin B 12 can be confounded by the presence of methionine, which may bypass the metabolic bottleneck caused by vitamin B 12 limitation in metE - E. coli and could affect the specificity of our assay. However, previous studies conducted on the E. coli 113-3 strain, another methionine auxotroph, showed that methionine must be 50,000 times more concentrated than vitamin B 12 to hinder vitamin B 12 quantification using the E. coli assay, which was significantly higher than the levels found in the mammalian tissues tested ( 12 ). Similarly, we did not observe unexpected E. coli B834 (DE3) growth supported by extracts derived from known vitamin B 12 non-producers ( Figure 2C ) or CeMbio collection strains, which were not predicted to produce vitamin B 12 ( Figure 3 ). Previous studies suggested that four strains in the CeMbio collection, C. piscis BIGb0172, P. berkeleyensis MSPm1, P. lurida , and O. vermis Myb71, are vitamin B 12 producers, based on the presence of vitamin B 12 biosynthetic pathway genes ( 29 ). Our analysis provided experimental evidence to support this. Interestingly, despite confirming that all four isolates are vitamin B 12 producers, we note that the levels of vitamin B 12 likely vary significantly, with P. berkeleyensis MSPm1 and C. piscis BIGb0172 producing significantly higher levels of vitamin B 12 compared to P. lurida Myb11 and O. vermis Myb71. These differences in vitamin B 12 content could be important for the growth of C. elegans and other organisms that directly rely on bacteria for vitamin B 12 . Conclusions Our results establish a high-throughput, straightforward, and cost-effective method for detecting and quantifying vitamin B 12 levels in biological samples. The simplicity, reproducibility, and sensitivity of the E. coli B834 (DE3) assay provide an important methodology for the research community working on vitamin B 12 . Our discovery of varying vitamin B 12 levels in the wild C. elegans microbiome makes a compelling case for further investigation into how differences in bacterial metabolites impact animal development. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials All data generated or analysed during this study are included in this published article [and its supplementary information files]. Competing interests The authors declare that they have no competing interests. Funding This work was supported by a UK Research and Innovation Future Leaders Fellowship [MR/S033769/1 and MR/X024261/1] awarded to Alper Akay, a Springboard Award from the Academy of Medical Sciences [SBF009\1005] and a Royal Society Research Grant [RGS\R1\231151] awarded to Matthew Sullivan. Katarzyna Hencel was funded by the University of East Anglia doctoral training programme. Author’s contributions Katarzyna Hencel, Matthew Sullivan and Alper Akay contributed to the conceptualisation, data analysis and manuscript writing. Katarzyna Hencel carried out the data acquisition and wrote the first draft of the manuscript. Alper Akay acquired the funding for the work. Supplementary Figure Legends Download figure Open in new tab Supplementary Figure 1. E. coli B834 was cultured in M9 minimal media with vitamin B 12 supplementations of 0.025 nM – 0.40 nM under standard carbon source input (0.4 % glucose; A ) or increased carbon source input (1.0 % glucose; B ). The data points are plotted with mean ± SEM from 3 biological replicates derived from 2 technical replicates corrected for blank readings. Download figure Open in new tab Supplementary Figure 2. (A) PCR validation of cbiA and cbiB deletions. For the cbiA gene deletion validation, primers A338 and A339 were used, with the wild type C. aquatica DA1877 genotype of 2672 bp and the length of 1274 bp of a successful cbiA mutant. For the cbiB mutation validation, primers A101 and A102 were used, with the wild type C. aquatica DA1877 genotype of 1182 bp and the length of 648 bp of a successful cbiB mutant. PCR validation was followed by the (B-C) Sanger sequencing validation using A338 and A102 primers for cbiA and cbiB genes, respectively, aligned against the reference genome of C. aquatica DA1877. The red dashed region of the sequencing profiles corresponds to the deleted portions of both genes and the lack of sequencing signal. The vertical dashed lines represent the cropped region of the deleted portions of the genes. (D - E) Vitamin B 12 quantification in C. aquatica DA1877 was performed by supplementing C. aquatica DA1877 extracts in (D) 10-fold and (E) 2-fold dilutions to E. coli B834. 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The functional repertoire contained within the native microbiota of the model nematode Caenorhabditis elegans . ISME J . 2020 Jan ; 14 ( 1 ): 26 – 38 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted April 08, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following High-throughput detection and quantification of vitamin B12 in microbiome isolates using Escherichia coli Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share High-throughput detection and quantification of vitamin B 12 in microbiome isolates using Escherichia coli Katarzyna Hencel , Matthew Sullivan , Alper Akay bioRxiv 2025.04.08.647760; doi: https://doi.org/10.1101/2025.04.08.647760 Share This Article: Copy Citation Tools High-throughput detection and quantification of vitamin B 12 in microbiome isolates using Escherichia coli Katarzyna Hencel , Matthew Sullivan , Alper Akay bioRxiv 2025.04.08.647760; doi: https://doi.org/10.1101/2025.04.08.647760 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7637) Biochemistry (17705) Bioengineering (13899) Bioinformatics (41968) Biophysics (21460) Cancer Biology (18603) Cell Biology (25526) Clinical Trials (138) Developmental Biology (13385) Ecology (19909) Epidemiology (2067) Evolutionary Biology (24326) Genetics (15614) Genomics (22513) Immunology (17741) Microbiology (40423) Molecular Biology (17193) Neuroscience (88645) Paleontology (667) Pathology (2835) Pharmacology and Toxicology (4825) Physiology (7647) Plant Biology (15160) Scientific Communication and Education (2046) Synthetic Biology (4302) Systems Biology (9825) Zoology (2271)