Cobalamin is present in cells of non-tuberculous mycobacteria, but not in Mycobacterium tuberculosis

Cobalamin (vitamin B12) is a structurally complex molecule that acts as a cofactor for enzymes and regulates gene expression through so-called riboswitches. The existing literature on the vitamin B12 synthesis capacity in Mycobacterium tuberculosis is ambiguous, while in non-tuberculous mycobacteria (NTM) is rather marginal. Here we present the results of our investigation into the occurrence of vitamin B12 in mycobacteria. For detection purposes, immunoassay methods were applied to cell lysates of NTM and M. tuberculosis clinical and laboratory strains grown under different conditions. We show that whereas vitamin B12 is present in cells of various NTM species, it cannot be evidenced in strains of differently cultured M. tuberculosis, even though the genes responsible for vitamin B12 synthesis are actively expressed based on RNA-Seq data. In summary, we conclude that the production of vitamin B12 does occur in mycobacteria, with the likely exception of M. tuberculosis. Our results provide direct evidence of vitamin B12 synthesis in a clinically important group of bacteria.


Results and discussion
Gene expression of vitamin B12 synthesis genes. We aimedto identify the genes involved in vitamin B12 synthesis in NTM included in this study (Table 1). We used whole-genome sequencing data and its annotation found in the major bioinformatics databases. The available data provided an incomplete indication of loci involved in the vitamin B12 biosynthesis pathway, as it is for M. tuberculosis. The precision of annotation, covering the entire extent of variability of proteins serving particular functions, is still to be developed. We used RNA-Seq data available at ENA Database to estimate gene expression through transcripts per million base pair (TPM) values for genes involved in vitamin B12 synthesis in M. tuberculosis, M. abscessus subsp. abscessus, and M. smegmatis (Table 2). TPM values inform about the level of basal transcription of genes, and are not to be confused with relative gene expression in different conditions. The average gene expression for M. abscessus and M. smegmatis was 201.88 ± 547.4 TPM and 147.33 ± 607.04 TPM, respectively. In comparison, the average expression of genes predicted to be involved in vitamin B12 synthesis was 94.943 ± 9.483 TPM and 76.669 ± 29.645 TPM, respectively. For M. tuberculosis we investigated gene expression level in cells grown in rich broth 20 , in medium supplemented with cholesterol 21 , and in human macrophages 22 . The above conditions' average gene expression was 256.02 ± 551.112 TPM, 256.01 ± 764.53 TPM, and 256.02 ± 1039.71 TPM, respectively. Simultaneously, the average expression of genes predicted to be involved in vitamin B12 aerobic synthesis was lower, 114.114 ± 77.666 TPM, 54.189 ± 35.772 TPM, 145.871 ± 159.664 TPM, respectively. Their overall expression level was comparable to DnaG primase, an essential protein involved in DNA replication (104.986 ± 2.321, 91.056 ± 42.023, and 118.236 ± 98.324, respectively) 23 .
Studies with Propionibacterium sp. showed the crucial role of cobA gene in regulating the level of synthesis of vitamin B12. Vitamin B12 was shown to regulate the cobA operon through a riboswitch in its 5′ untranslated region (5′ UTR) 24 . Similarly, M. tuberculosis contains a PPE2-cobQ1-cobU operon, containing vitamin B12 synthesis genes and controlled by a riboswitch. Taken the ubiquity of vitamin B12 riboswitches across Prokarytotes, the mechanisms where the level of vitamin B12 synthesis genes seem to be controlled by the synthesis product might be common 25 . Presented results show that cobQ1 and cobU of M. tuberculosis are actively expressed in a rich broth and in the presence of cholesterol. Expression of cobQ1 was not observed in macrophages. The level of reading coverage of the mycobacterial genome is relatively low. We assume that the low coverage results from natural technical difficulties of isolating mycobacterial RNA from the Eukaryotic cells that have RNA of their own 26 . Since reads of cobU are present, we suspect that the absence of cobQ1 reads in macrophages is due to too low coverage.
Vitamin B12 concentration in non-tuberculous mycobacteria. We measured the concentration of vitamin B12 per mg of protein in cell lysates obtained from 7H9 medium cultures of various species of NTM spread across the phylogenetic tree (Fig. 3A) 27 19 . When vitamin B12 concentration was normalized to protein content, we detected a higher concentration of vitamin B12 in mycobacteria than it was previously detected in P. aeruginosa. There, analyses by HPLC-MS detected from 0.32 to 3.72 ng of vitamin B12 per mg of protein, depending on culture conditions and strain 28 .
There are different approaches to the normalization of vitamin B12 concentration in bacteria. To further compare our results with other bacterial species, we also normalized our data regarding vitamin B12 concentration to ml of culture (Fig. 3B). When calculated in such a way, we obtained from 0.049 to 1.2 ng of vitamin B12 per ml of culture. In comparison, Pseudomonas freudenreichii produced from 20 to 125 ng of vitamin B12/ ml of culture, depending on culture conditions 29 . B. megaterium produced from 0.26 ng/ml of culture to 204 ng/ ml of culture, also depending on the culture conditions. Due to relatively low concentration of vitamin B12, expensive growth media, long culture time, and difficulties to disrupt the cells, we conclude that mycobacteria are not attractive alternative producers of vitamin B12 at the industrial scale.
Importantly, we show that NTM can produce vitamin B12, and synthesis is shared across the phylogenetic tree. The sensitivity of the immunoassay detection was suitable for the detection of vitamin B12 in mycobacterial cells. This observation is an important reference point for results obtained for M. tuberculosis. We observed up to a twofold difference in the level of vitamin B12 synthesis across distinct strains of the same species. Strain variability in cobalamin concentration was observed previously in Pseudomonas aeruginosa, where the concentration of vitamin B12 ranged from 0.84 to 3.72, hence changed four-fold, depending on a strain 28 .

Salvage pathway and transport
bacA Cobalamin transporter

Anaerobic pathway
cbiA CbiA domaincontaining protein Putative amidotransferase similar to cobyric acid synthase

Salvage pathway and transport
bacA Cobalamin transporter Strain variability in the level of vitamin B12 production is important in the context of M. tuberculosis. Data presented in previous manuscripts suggested indirectly that certain strains of M. tuberculosis may be capable of cobalamin synthesis, while others are probably not 14,31 . As in other species, mycobacteria do show a certain spread in the level of vitamin B12 synthesis that probably can be attributed to the genetic background rather than the environmental factors or stage of the growth.

Urpoporfirynogen III pathway
Increased concentration of vitamin B12 in mycobacterial cells under starvation results from accumulation rather than increased production. In our previous study, we showed that cells of M. smegmatis grown in a medium deprived of nutrients contain an approximately eightfold amount of vitamin B12 when compared to cultures grown in a rich broth. An increase in vitamin B12 concentration was also observed in stationary phase cultures 17 . A similar observation was made in P. aeruginosa. There, vitamin B12 concentration increased from non-detectable during exponential growth to 0.32-0.67 ng/mg of protein in stationary phase cultures, depending on a strain. The concentration further increased up to 3.72 in conditions of continuous-flow growth 28 .
Here, we show that the reason behind the increased concentration of vitamin B12 in starved cells of M. smegmatis mc 2 probably results from accumulation rather than increased synthesis. We estimated the relative gene expression of genes involved in cobalamin synthesis in starved cells compared to cells in the logarithmic phase (Fig. 4). We observed that the expression of genes involved in vitamin B12 synthesis was either constitutive (0 to 1-fold change in relative expression to sigA) for cobG, cobL, cobO and cobD or repressed (> 3-fold change) for cobU and cobN.
Accumulation of vitamin B12 in starved cells and old cultures of M. smegmatis is important in the context of cobalamin detection in M. tuberculosis. M. smegmatis is a model organism for studying the biology of mycobacteria, including M. tuberculosis 32 . Bacteria of the same phylogenetic order are likely to maintain the same biological pathways and mechanisms. Indeed, increased expression of cobalamin synthesis genes was reported in dormant cultures of M. tuberculosis 16 . Therefore, if cobalamin was to be present in the cells of M. tuberculosis, it was more likely to be identified in prolonged, starved, or dormant cultures.
Lack of observable vitamin B12 production in M. tuberculosis. We tested the contents of cells of M. tuberculosis for vitamin B12 by immunoassay (Table 3). We included laboratory strain of M. tuberculosis H37Rv and five clinical strains of M. tuberculosis, here grouped into a group of "clinical strains". As a negative control strain, we used M. tuberculosis deficient in cobIJ gene. Predicted function of cobIJ is precorrin-2 C20-methyltransferase/precorrin-3B C17-methyltransferase. The gene product is required at the early stage of vitamin B12 synthesis (Fig. 1).
We screened M. tuberculosis cell lysates derived from cultures grown in various conditions. The growth conditions aimed to mimic the environments that can be found during M. tuberculosis infection cycle. All cultures were hemD Next, we wanted to see if M. tuberculosis might rely on substances widely present in the host to produce vitamin B12. We supplemented the growth medium with uroporphyrinogen III, which is a precursor of heme in the human body and a precursor of vitamin B12 in bacteria (Fig. 1). We tested cell lysates from logarithmic phase cultures, stationary phase cultures, persister cell cultures, and hypoxic cultures. ELISA immunoassay detected

M. tuberculosis metE promoter responds to vitamin B12 concentration present in the host.
We used the GFP reporter system to see if the cells of M. tuberculosis could respond to vitamin B12 concentration found within the host (Fig. 5). Vitamin B12 concentration in the human body is between 0.2 and 0.9 μg/ml. We constructed a series of mutants, H37Rv::attB + rsB12, ∆bacA::attB + rsB12, ∆cobIJ::attB + rsB12, carrying the gene of GFP under the control of the metE promoter, controlled by a vitamin B12-dependent riboswitch. In our model, the presence of vitamin B12 in the cells prevents translation of gfp transcript, which results in diminished fluorescence. Of note, distinct clones of the same cell lines showed a different level of basal fluorescence without supplementation of medium without vitamin B12. Therefore, the fluorescence level could not be reliably compared between different cell lines due to the distinct basal expression of GFP in the clones. However, green fluorescence levels could be relatively compared within one clone of the cell line when considering different concentrations of vitamin B12 in the growth medium. We tested various concentrations of vitamin B12. We observed that supplementation of the growth medium with vitamin B12 gradually diminished gene expression of the green fluorescence protein of M. tuberculosis H37Rv and ∆cobIJ from 100 to 23.93% and 23.70%, respectively. In turn, the green fluorescence expression of ∆bacA was not affected, and it remained constant at approximately 100%. Similarly, the autofluorescence level was constant for the control strain M. tuberculosis H37Rv, which lacked the reporter system. M. tuberculosis H37Rv GFP expression diminished to 70.29% in the presence of 0.5 μg/ml of vitamin B12 (p = 0.01, t = 6.64, df = 3). Hence, M. tuberculosis metE promoter is responsive to vitamin B12 concentration found in the human body. Our results confirm the role of BacA as the transporter of vitamin B12 33 . Further, our results indirectly confirm the lack of vitamin B12 production in M. tuberculosis H37Rv, because the wild type strain and the knock-out strain similarly showed a decrease in fluorescence corresponding to increasing concentration of supplemented vitamin B12. Previous reports suggested that the ability to synthesize vitamin B12 by M. tuberculosis was restricted in M. cannetti like ancestor 9,10 . M. tuberculosis is an obligate pathogen with possible access to vitamin B12 from the host. It is, therefore, possible that the genes involved in vitamin B12 synthesis in the genome of M. tuberculosis Figure 3. Cobalamin concentration in cell lysates of non-tuberculous mycobacteria. Cobalamin was detected in cell lysates of non-tuberculous mycobacteria by immunoassay. Cells were grown in 7H9 medium supplemented with OADC, Tween 80, and CoCl 2. We cultured cells until the suspension reached OD 600 = 1. Next, cells were harvested and washed with fresh medium without supplements to remove residual medium proteins from the surface. The pellet was re-suspended in Tris buffer and disrupted by beat-beating. The suspension was spinned. We used supernatant to estimate the concentration of vitamin B12 and protein content. Results were obtained from three independent cultures; each lysate was analyzed in two technical replicates. www.nature.com/scientificreports/ are remnants from a more independent ancestor. The genes of the vitamin B12 biosynthesis pathway in Mycobacterium leprae, another obligate pathogen of Mycobacterium genus, evolved into pseudogenes 10 . The most probable explanation is that M. tuberculosis does not synthesize vitamin B12 anymore. However, there was still not enough time since the abrogation of the pathway to accumulate mutations that would entirely degrade the pathway, impede the expression of genes and convert them into pseudogenes. It seems that the disruption of the pathway might have taken place relatively recently, as cobF encoding region was found in two M. tuberculosis strains found in the African Great Lakes region, representing Lineage 8 of M. tuberculosis complex 31 . To be precise, it cannot be excluded that M. tuberculosis does synthesize vitamin B12, but the level of vitamin concentration is undetectable by the immunoassay we used. Finally, it remains to be established whether M. tuberculosis is able to synthesize cobamides other than vitamin B12 34 .

Summary
We conclude that mycobacteria are generally capable of vitamin B12 synthesis, with the likely exception of M. tuberculosis. Our results are direct evidence of vitamin B12 production in these clinically important group of bacteria.
We tested several types of cultures of M. tuberculosis for vitamin B12 concentration. Logarithmic phase cultures of M. tuberculosis were grown in 7H9 medium supplemented with OADC, Tween80, and cobalt chloride until they reached OD600 = 0.8. Stationary phase cultures were carried out in the same medium, and they were collected after 15 days of culture. Acidified cultures (pH 5.7) were carried out in 7H9 broth, supplemented with 5% bovine serum albumin, fraction V, 2% glucose, and 0.85% NaCl, Tween 80, and cobalt chloride. They were collected after 1 week of culture. Starved cultures were carried out in 7H9 broth supplemented with Tween 80, 0.5% glycerol, and cobalt chloride. They were collected after one week of culture. Hypoxic cultures were carried out as previously described 37 . In brief, starter cultures of M. tuberculosis grown on Dubos medium supplemented with OAD and cobalt chloride were tightly locked in flasks with 0.25 headspace ratio and cultured at 37 °C on a shaker for six weeks. Catalase is not recommended for use in hypoxia experiments because it influences redox balance, and redox stress is an important stress factor of hypoxia 38 . Methylene blue was added to control cultures as an indicator of oxygen depletion. After this time, the flasks were opened, cultures were spinned down and washed three times with 7H9 medium. A sample of the culture was plated as viability control, while the rest was lyzed. For persister cultures, we used a medium deprived of K+, as previously described 16 . Here, starter cultures grown on Sauton medium were spinned down and re-suspended in Sauton medium deficient in K+ supplemented with cobalt chloride. After two weeks, rifampicin was added to cultures at 5 μg ml −1 and the culture continued for the next four weeks at 37 °C.
Cloning strategy. All molecular cloning was performed in E. coli T10. Knock-out mutants of mycobacteria were obtained by the method of gene replacement through homologous recombination (Tables 5, 6; Fig. 6; Supplementary Figs. 1 and 2) 39 . Briefly, sequences flanking desired deletion were amplified by PCR. We used AccuPrime Pfx High Fidelity Polymerase (Invitrogen), and genomic DNA of M. tuberculosis H37Rv for the reaction. PCR products were introduced into pJET1.2 plasmid (Thermo Fisher Scientific) and sequenced. Following confirmation of cloning of proper sequence, we cut out flanking sequences using restriction enzymes, and sequentially introduced them into p2NIL plasmid, together with marker genes from pGOAL17. Plasmids were transformed into M. tuberculosis H37Rv thru electroporation. The cells underwent gene replacement by allelic exchange as described previously 39 . Similarly, episome plasmid containing green fluorescence protein (GFP) gene under the control of the riboswitch of M. tuberculosis metE gene was constructed with a similar procedure. We started with PCR amplification of products on genomic DNA of M. tuberculosis H37Rv and pJAM plasmid carrying gfp. Subsequently, we introduced sequences to pJET1.2, and we confirmed proper cloning by sequencing. Next, we used restriction digestion to cut out the sequences, and we introduced them into pMV306 episome plasmid. M. tuberculosis H37Rv and its derivative strains were transformed by electroporation.
Vitamin B12 ELISA. Bacterial cultures were spinned down, washed with fresh medium without supplements, spinned down again, and re-suspended in 0.01 M TRIS pH 7.5. The mixture was transferred to disruptor eppendorfs. Cells were disrupted twice using the MP disruptor system with the Quick prep adapter (MP Biomedicals) and 0.1 mm silica spheres (45 s, 6.0 m/s with 5 min intervals). Samples were spinned down, and lysates were transferred to new eppendorfs. As a principal, we normalized the results regarding vitamin B12 concentration to protein concentration in cell lysates. We wanted to avoid errors resulting from a different level of difficulty to disrupt mycobacterial cells of different species. Protein concentration in lysates was measured using Bradford reagent (BioShop) and estimated with a standard curve. In order to achieve a sufficient detection limit of vitamin B12, we only used the lysates that contained at least 0.5 mg of protein per ml, preferably between 1 and 2 mg of protein per ml.
Vitamin B12 ELISA (Demeditec) was performed according to manufacturer instructions. The test is based on the principle of the competitive enzyme-linked immunosorbent assay. The surface of a microtiter plate was covered with an antibody directed against vitamin B12 by the manufacturer. Samples and standards were mixed with a vitamin B12-peroxidase conjugate in the wells of the microtiter plate. Both enzyme-labeled and free vitamin B12 competed for the antibody binding sites. After one hour of incubation at room temperature, the wells were washed to remove the unbound material. A substrate solution was added, resulting in the development of a blue color. The color development was inhibited by the addition of a stop solution, and the color turned yellow. The yellow color was measured photometrically at 450 nm. The concentration of vitamin B12 was indirectly proportional to the color intensity of the test sample.
For each condition, we analyzed lysates from three independent cultures. Each sample was analyzed in duplicate wells, as recommended by the producer of the immunoassay. The minimum detection level of vitamin B12 was settled at 1 ng/ml based on our previous observations 17 , and all samples below this level were considered negative for vitamin B12. For purposes of enabling comparison with the results obtained from other species of bacteria, our results were also normalized to ml of culture.

Flow cytometer analysis. Samples of cultures were analyzed on the flow cytometer Guava EasyCyte Flow
Cytometer with High Power Blue Laser (Merck) suitable for detection of bacteria. Unstained control samples were diluted to reach a concentration of 400-800 cells/μl. Cell suspensions were first run through the flow cytometer to set a population gate around the bacteria by using the forward-scatter versus side-scatter parameters. Next, the voltages in the green fluorescence channel were adjusted so that the fluorescence histogram of the unstained bacteria appeared within the first compartments of the logarithmic scale of fluorescence. Ten   21 and in human THP-1 derived macrophages three days post-infection. Each experiment contained data for three replicates. Raw sequences were uploaded and processed with Geneious Prime 2021 (Biomatters, New Zealand). Reads were mapped to M. tuberculosis H37Rv accession number NC_000962 using Bowtie2 Geneious plug-in 42 . Gene expression analysis, through estimation of transcripts per kilobase million (TPM), was performed with Geneious.