Cobalamin production by Lactobacillus coryniformis: biochemical identification of the synthetized corrinoid and genomic analysis of the biosynthetic cluster

Background Despite the fact that most vitamins are present in a variety of foods, malnutrition, unbalanced diets or insufficient intake of foods are still the cause of vitamin deficiencies in humans in some countries. Vitamin B12 (Cobalamin) is a complex compound that is only naturally produced by bacteria and archea. It has been reported that certain strains belonging to lactic acid bacteria group are capable of synthesized water-soluble vitamins such as those included in the B-group, as vitamin B12. In this context, the goal of the present paper was to evaluate and characterize the production of vitamin B12 in Lactobacillus coryniformis CRL 1001, a heterofermentative strain isolated from silage. Results Cell extract of L. coryniformis CRL 1001, isolated from silage, is able to correct the coenzyme B12 requirement of Salmonella enterica serovar Typhimurium AR 2680 in minimal medium. The chemical characterization of the corrinoid-like molecule isolated from CRL 1001 cell extract using HPLC and mass spectrometry is reported. The majority of the corrinoid produced by this strain has adenine like Coα-ligand instead 5,6-dimethylbenzimidazole. Genomic studies revealed the presence of the complete machinery of the anaerobic biosynthesis pathway of coenzyme B12. The detected genes encode all proteins for the corrin ring biosynthesis and for the binding of upper (β) and lower (α) ligands in one continuous stretch of the chromosome. Conclusions The results here described show for the first time that L. coryniformis subsp. coryniformis CRL 1001 is able to produce pseudocobalamin containing adenine instead of 5,6-dimethlbenzimidazole in the Coα-ligand. Genomic analysis allowed the identification and characterization of the complete de novo biosynthetic pathway of the corrinoid produced by the CRL 1001 strain.


Background
Lactic acid bacteria (LAB) are a heterogeneous group of bacteria extensively used as starter cultures for the fermented foods development. Because of their metabolism, this group of bacteria can improve the safety, shelf life, nutritional value, flavour and overall quality of fermented products. Some strains have shown to exert a large range of beneficial properties in humans, and they are frequently used as probiotic microorganisms. In addition, certain LAB strains are able to produce and release compounds with biological activity in foods, sometimes referred as nutraceuticals [1]. These micronutrients are used as a cofactor in numerous enzymatic reactions, as it is the case of vitamin B 12 .
Conceptually, the definition of vitamin B 12 generally describes a type of cobalt corrinoid, belonging to the cobalamin (Cbl) group. On the other hand, vitamin B 12 is the form of the vitamin obtained during process of industrial production but this form not exists in the nature [2]. Naturally, this compound is found as desoxyadenosilcobalamin (coenzyme B 12 ), methylcobalamin or pseudocobalamin, among other forms. Concerning the structure, the cobalamin molecule present 3 parts main: (i) the central corrinic ring with the four ligands of a cobalt ion, (ii) a superior (or beta) ligand that is attached to adenosyl o methyl group, and (iii) the lower ligand (or alfa), usually dimethylbenzimidazole (DMB). It has been described that in some anaerobic bacteria, the adenine and other ligands can replace DMB giving as a result pseudocobalamin (pseudo-B 12 ) and other active cofactors [3]. This soluble compound is one of the most complex non-polymeric macromolecules produced in the cell and only a reduced group of bacteria and archaea are able to synthesize it. The bacteria present in the rumen of cattle, sheep and other ruminants are the responsible of its synthesis. Humans do not have such microbiota in their large intestine and must absorb the coenzyme from natural sources such as meat, fish, eggs, and pharmaceutical products [4]. A diet sufficient in vitamin B 12 is essential to prevent severe pathologies (megaloblastic anaemia, pancytopenia, peripheral neuropathy, increased risk of myocardial infarction and stroke, others) some of which are irreversible [5][6][7][8]. Thus, the use of vitamin-producer bacteria appears as an appropriate alternative to produce innovative foods with added nutritional value [9]. However, the production of vitamin B 12 is strain specific and an unusual trait present in certain strains of the Lactobacillus genus. The production of this metabolite by Lactobacillus reuteri strains has been described for the first time by us and confirmed later by others authors [10][11][12][13]. Moreover, we validated that the corrinoid (pseudo-B 12 ) produced by L. reuteri CRL 1098 is effective in preventing the hematological and neurological pathologies (anaemia, growth retardation, others) caused by a diet deficiency in vitamin B 12 in a combined animal model (pregnant mice and their offspring) [14,15]. More recently, De Angelis et al. [16] described the production and the genetic organization of the complete de novo biosynthetic pathway of vitamin B 12 in Lactobacillus rossiae DSM 15814. Among other lactobacilli, previous evidence suggested that two strains of L. coryniformis (CECT 5711 and strain 394) are capable of producing the antimicrobial compound reuterin [17,18], an enzymatic pathway that involves a cobalamin-dependent enzyme, the glycerol dehydratase [19]. No further analysis related to vitamin B 12 production was carried out in those studies. Based on this previous evidence, the purpose of the present study was to evaluate the vitamin B 12 production in L. coryniformis CRL 1001, a heterofermentative strain isolated from silage. Firstly, the compound with cobalamin activity was isolated and biochemically characterized. Next, the genome of the CRL 1001 strain was sequenced by whole genome shotgun (WGS) sequencing strategy and the identification of putative biosynthetic operon involved in the synthesis of the corrinoid was performed by in silico analysis. Finally, a detailed comparative study of the cobalamin gene cluster of L. coryniformis CRL 1001 with those of previously known vitamin-producer strains was performed. The results of this research provide the first evidence of vitamin B 12 synthesis by a L. coryniformis strain.

Results and discussion
Cobalamin production by L. coryniformis CRL 1001 In order to evidence the production of cobalamin by L. coryniformis CRL 1001, we use S. Typhimurium AR 2680 (metE cbiB) [20] as indicator strain, grown in minimal medium without vitamin B 12 according the methodology described by Taranto et al. [12]. The results of the bioassay showed that the cobalamin requirements of the indicator strain were corrected when Cell Extracts (CE) of L. coryniformis CRL 1001 were added; the halo diameter obtained was similar to that obtained with a solution of standard cyanocobalamin (CN-Cbl). On the other hand, no growth was detected when the CE from Lactobacillus plantarum ATCC 8014 (negative control strain) was used. These results would indicate that L. coryniformis CRL 1001 is able to produce compounds with cobalamin activity.
To identify the compound with cobalamin activity produced by L. coryniformis CRL 1001, the CE was analysed by RP-HPLC. The UV-DAD spectra data revealed the presence of two main peaks that showed similar characteristics with the spectrum obtained for the peak belonging to the CN-Cbl standard. The Retention Times (RT) of these peaks were slightly lower (23.77 min and 24.20 min) to the RT of standard solution of CN-Cbl (24.98 min) (Fig. 1). However, when these peaks were collected and analysed using the bioassay, they showed the same ability of vitamin B 12 complementation than the CN-Cbl standard. For this reason, we continued with the identification of both peaks.
Characterization of the corrinoid produced by L. coryniformis CRL 1001 Corrinoid compounds separated by HPLC were analysed by Liquid chromatography-electrospray ionization/tandem mass spectrometry (LC/ESI-MS/MS). The mass module parameters were fine-tuned and optimized with commercial standard of CN-Cbl. The transition 678.3 (m/z) [M + 2H + ] ++ to 358.7 (m/z) corresponds to the lower ligand, whose DMB is the aglycon attached to ribofuranose 3-phosphate whereas the transition 678.3 (m/z) [M + 2H + ] ++ to 146.9 (m/z) corresponds to the base DMB. Data transition for pseudo-B 12 were estimated and calculated according the bibliography as follows: 672.5 (m/z) [M + 2H + ] ++ to 347.8 (m/z); this transition corresponds to the lower ligand where adenine is the aglycon attached to ribofuranose 3-phosphate, and the transition 672.5 (m/z) [M + 2H + ] ++ to 135.9 (m/z) corresponds to the base adenine [21]. Both transitions were sought in the MS and MS/MS spectra of the peaks with cobalamin activity.
The MS spectra of the active peaks obtained by HPLC indicated that a double charged ion with an approximately m/z of 673 [M + 2H + ] ++ was prominent in the two spectra (Fig. 2). The MS/MS spectrum indicated that the dominant ions with a value approximate m/z 347.8 were attributable to the substitution in the lower ligand where the adenine is the aglycon attached to ribofuranose 3-phosphate.
The MS showed that the molecular species (corrinoid) synthesized by L. coryniformis CRL 1001 corresponds mainly to Coα-[α-(7-adenyl)]-Coβ-cyanocobamide, commonly known as pseudo-B 12 , an analogue with a structure very similar to vitamin B 12 . In this corrinoid, the DMB moiety is substituted by adenine as lower ligand. This compound is a physiologically important form of the vitamin B 12 in various microorganisms [22].
The concentration of pseudo-B 12 produced by L. coryniformis CRL 1001 was calculated indirectly using a standard curve made with different concentrations of a commercial CN-Cbl. The area of the peaks obtained in the chromatograms corresponds to approximately 0.94 μg mL −1 of pseudo-B 12 produced.
Genome identification of vitamin B 12 cluster in L. coryniformis CRL 1001 The genome sequence of L. coryniformis CRL 1001 consisted on 133 contigs with a total size of 2,829,178 base pairs and an average GC content of 42 %. The structural and functional annotation performed with the Rapid Annotations using Subsystems Technology (RAST) server [23] allowed us to identify 3341 coding sequences (CDS) and 82 structural RNAs (58 tRNAs) [23]. Additionally, it was observed that 332 subsystems that include the 44 % of the identified CDS are represented in the chromosome.
In silico genomic analyses revealed the presence of 32 open reading frames (ORFs) related to the coenzyme B 12 production (cbi, cob, hem and cbl gene cluster) located immediately adjacent to the pdu operon (22 ORFs) which encodes the enzymes necessary for the use of glycerol or propanediol as carbon source (Fig. 3). As described in other species, the close association of these two gene clusters is probably a reflection of the requirements of cobalamin or its corrinoid derivatives for the use of propanodiol [24]. Almost all predicted genes (pdu, cbi, cob and hem) are in the same orientation, except for pocR, which encodes for a transcriptional regulator with 45 % identity and 69 % positive matches at the amino acid level, with its homolog in L. reuteri JCM 1112 [25]. Interestingly, the two clusters (pdu and cbicob-hem) have a few intergenic regions and their average G + C content (52 %) differ from that of the draft genome sequence of L. coryniformis CRL 1001 (42 %). As described by Morita et al. [10] for L. reuteri JCM 1112, this data could explain the existence of a genomic island acquired through horizontal gene transfer.
Among pdu genes, pduC, pduD and pduE were found. These genes encode for the three subunits of the glycerol dehydratase, an enzyme that requires cobalamin as a cofactor for performing it catalytic activity [19]. Additionally, a gene coding for the L-threonine kinase PduX, was localized. This protein, described in Salmonella enterica, transfers a phosphoryl group to a free L-threonine and it is involved in de novo synthesis of Adenosyl cobalamin and the assimilation of cobyric acid (Cby) [26].
Downstream of the pdu operon, seventeen cbi genes (cbiA, B, C, D, E, T, F, G, H, J, K, L, M, N, Q, O, and P) and cysG gene were in silico identified. The proteins encoded by these genes are required in most of the reactions that take place in the cell during the corrinoid ring synthesis of the cobalamin molecule [27].
Regarding cob genes cobA, D, U, S and C were found. cobA encodes an adenosyltransferase, a binding protein of adenosyl group as β-ligand [28]. The gene cobD encodes an L-threonine-O-3-phosphate descarboxylase that is involved in the amino-propanol arm synthesis [29] while cob U, S and C encode the proteins that participate in the attachment of the amino-propanol arm and in the assembly of the nucleotide loop that connects the lower cobalt ligand to the corrinoid ring [30].
Finally, downstream of the cbi and cob genes, five hem genes (hem A, C, B, L and D) were detected in a similar way as observed for other microorganisms that produce cobalamin anaerobically [31]. The proteins encoded by these genes are involved in the first steps of the cobalamin biosynthesis, the synthesis of uroporphyrinogen III from L-glutamyl-tRNA(glu). At the same time, the hemD/cobA gene, encoding a single polypeptide with both uroporphyrinogen III synthase and methyltransferase activity was detected [32].
Interestingly, the genome of L. coryniformis CRL 1001 lacks cobT gene, contrary to that observed in the cobalamin biosynthetic cluster of L. reuteri CRL 1098 [11]. Instead, two cbl genes were detected, cblS and cblT, encoding for a α-ribazole phosphoribosyl kinase and a α-ribazole transporter, respectively. These results might suggest an alternative pathway for α-ribazole salvaging and the α-ribazole-P synthesis in L. coryniformis CRL 1001, as it is described in Listeria [33].
In silico comparative analysis of the biosynthetic cluster of vitamin B 12 The cobalamin biosynthetic gene cluster has been identified in a few Lactobacillus species, including L. reuteri [11] and more recently in L. rossiae [16]. Both strains are obligate hetero-fermentative LAB [34]. L. coryniformis CRL 1001 is the first facultative heterofermentative strain described as cobalamin producer.
Comparative genomic studies among L. coryniformis CRL 1001, L. reuteri DSM 20016 and L. rossiae DSM 15814 strains evidenced a conserved genetic organization of the coenzyme B 12 biosynthetic genes. In these three strains, the cbi-cob-hem cluster is adjacent to the pdu cluster (Fig. 4). However, contrasting other cobalamin producer strains, this two gene sets (pdu and cbi-cob-hem) have an opposite orientation in the L. rossiae genome. Morita et al. [10] suggested that both gene cluster might have been acquired by horizontal transfer and inserted independently in these genomes.
As previously described for cobalamin producer anaerobic strains, the hem genes were located among the cbicob genes in all the organisms under comparison.
The cblS and cblT genes identified in the genome of L. coryniformis were also detected in the genome of L. rossiae where they have an identical localization in the cbicob-hem cluster. However, these genes are absent in the genome of L. reuteri. The cobT gene, encoding a Nicotinate mononucleotide (NaMN): base phosphoribosyltransferase, has only been described in this latter specie for the strains L. reuteri CRL 1098 (AY780645.1), L. reuteri JCM 1112 (NC_010609.1) and L. reuteri DSM 20016 (NZ_AZDD00000000.1).
Until now, among the Lactobacillus strains described as cobalamin producers, L. coryniformis CRL 1001 is the only strain harbouring the gene encoding for PduX on its genome (Fig. 4).
A comparative analysis between the deduced amino acid sequences of each ORF included in the vitamin B 12 biosynthetic cluster of L. coryniformis CRL 1001 and those from L. rossiae, L. reuteri, Listeria sp. and Salmonella sp. using the BLASTP algorithm, was performed. We found that most of L. coryniformis CRL 1001 proteins have the highest percentage of identity with those identified in L. rossiae strain (Table 1), with the exception of CobD and PduX, which were more similar to Listeria proteins. This result indicates that the aminopropanol phosphate synthesis from L-threonine in L. coryniformis CRL 1001 is similar to that performed by Listeria strains, previously described by Fan et al. [26]. These results suggest an alternative vitamin B 12 biosynthetic pathway in this genus.

Conclusion
In this work, we demonstrated for the first time that L. coryniformis subsp. coryniformis CRL 1001 is able to produce corrinoids with cobalamin activity. The chemical characterization of the molecule isolated from CRL 1001 CE using HPLC and mass spectrometry show that the majority of corrinoid produced is pseudocobalamin that has adenine like Coα-ligand instead 5,6-dimethylbenzimidazole. This molecule is one of the forms of coenzyme B 12 occurring in nature. Genomic studies revealed the presence of the complete anaerobic biosynthetic pathway of coenzyme B 12 . The comparative analysis of the cobalamin gene cluster of L. coryniformis CRL 1001 with those present in strains previously described as vitamin producers, revealed that this strain bears the genes cblS-cblT only present in the genome of L. rossiae DSM 15814. Moreover, pduX gene has not been detected in another strain of the Lactobacillus genus. The results of this work provide the first evidence of vitamin B 12 synthesis by a L. coryniformis strain.

Methods
Strains, media and culture conditions L. coryniformis CRL 1001, a strain originally isolated from silage, belongs to the CERELA-CONICET culture collection. The CRL 1001 strain was grown in Man-Rogosa-Sharpe (MRS) broth and Vitamin B 12 Assay Medium (Merck, Germany) overnight at 37°C without shaking. S. Typhimurium AR 2680, a strain with mutations in the genes metE and cbiB, was used as indicator strain in bioassays for cobalamin determination. This strain was grown in Luria-Bertani (LB) broth at 37°C with aeration. To perform the bioassay, the indicator strain was grown in minimal A medium (NaCl, 0.5 g L −1 , Na 2 HPO 4 , 6 g L −1 ; KH 2 PO 4 , 3 g L −1 , NH 4 Cl 1 g L −1 ; glucose, 4 g L −1 ; MgSO 4 , 2 mM, CaCl 2 0.1 mM). As negative control, L. plantarum ATCC 8014 was employed.

Preparation of cultures and cell-extracts
The production of corrinoid type-cobalamin by L. coryniformis CRL 1001 was detected as following: a culture was inoculated duplicated in vitamin B 12 -free assay medium, grown at 37°C for 16 h and subcultured three times in the same medium before use. Cell Extracts (CE) were prepared from 100 mL of culture grown for 24 h; after harvesting (8000 × g, 5 min), cells were washed in 10 mL of 0.1 M phosphate buffer pH 7.0 twice and resuspended in 10 mL of the same buffer and were fasted overnight at 2°C. Cells were harvested (8000 × g, 5 min) and resuspended in 10 mL of extraction buffer (0.1 M Na 2 HPO 4 , pH 4.5 reached with, 0.005 % KCN). The cell suspension obtained was autoclaved at 120°C for 20 min, the pellet was separated by centrifugation (10,000 × g, 20 min), and the supernatant was passed over a solid-phase extraction (SPE) column C18-E (Phenomenex) with 500 mg particle size and 6 mL reservoir volume; the column was previously activated with 6 mL of methanol HPLC degree (Loba Chemie, India). The column was washed with 2 volumes of ultra-purified water twice to remove salts and other hydrophilic contaminants. The potentials corrinoid was eluted with 1

Cobalamin detection
To demonstrate the production of cobalamin by L. coryniformis CRL 1001, a bioassay using S. Typhimurium AR 2680 as indicator strain was performed. The CE from L. coryniformis CRL 1001 was examined for its ability to correct the cobalamin requirement of the AR 2680 strain in minimal medium. For this purpose, S. Typhimurium AR 2680 cells were grown for 16 to 18 h in LB medium; the active culture was seeded onto minimal agar medium specific for this strain. Four wells were performed in each agar plate. Fifteen microliters of CE from L. coryniformis CRL 1001 (dilutions 1 10 −1 and 1 100 −1 ) were loaded in each well and incubated 24 h under anaerobic conditions at 37°C. As comparative standard of

Isolation and characterization of the corrinoid
To isolate the corrinoid produced by L. coryniformis CRL 1001, the CE was loaded onto a C 18 solid-phase 5 μm extraction column (250 × 4.6 mm) (Shimadzu Shim-Pack VP-ODS) and purified by reverse-phase (RP)-HPLC using a LC_20 AT system automated gradient controller with a SIL-20ª HT autosampler and a SPD-M20A diode array UV-visible detector at 361 nm (Shimadzu Corporation, Kyoto, Japan). The column was thermostated at 25°C. The mobile phases was filtered through a 0.22 μm membrane filter and vacuum degassed prior to use. The mobile phases were prepared with ultra-purified water with 0.1 % formic acid (Solvent A) and methanol with 0.1 % (v/v) formic acid (Solvent B). The protocol used was described by Yu et al. [35] with some modifications. The following mobile phase gradients (Solvent B) were used: 0-2 min (20 %); 2-5 min (35 %); 5-22 min (100 %); 22-26 min (100 %); 26-36 min (20 %); and 36-45 min (5 %); this step was followed by the final passage of Solvent B at 5 % for re-optimization of the parameters in the module HPLC. The volume of injection was 100 μl. The HPLC was operated in a constant flow mode and the flow rate was kept at 0.5 ml min −1 .
The peaks with RT close to the RT of CN-Cbl standard (Sigma-Aldrich, cod. V2876) (concentration = 5 μg mL −1 ) were collected and concentrated to dryness under vacuum at 30°C. Then, the peaks were dissolved with methanol 50 % and stored in the dark at -20°C until use. The vitamin B 12 activity of each collected peak at 1.10 −1 and 1.100 −1 dilutions was detected using the bioassay with the Salmonella strain. Peaks with vitamin B 12 activity positive were analysed by mass spectrometry.
The active peaks collected from the RP-HPLC were analysed using Ultimate 3000 RSLC Dionex -Thermo Scientic with a C 18 column 50 × 2.1 mm that contained 1.9 micron particles (Hypersil-GOLD). The solvents for Liquid Chromatography were solvent A (ultra-purified water with 0.1 % formic acid) and solvent B (methanol with 0.1 % formic acid). The flow rate was 0.2 mL min −1 and the injection volume was 10 μL. Chromatography was in gradient, and the solvent composition was chosen such that the compound of interest eluted 8-9 min after injection. The mobile phase gradients (solvent B) were maintained during a 35 min run: 0-2 min (5 %); 2-5 min (35 %); 5-22 min (100 %); 22-24 min (100 %); 24-28 min (5 %); and 28-35 min (5 %), this step was followed by 50 % of solvent B for re-optimization of the parameters in the module HPLC coupled with Mass. A single peak for each sample was eluted and the area of the signal was used for quantitative analysis. Positive-ion MS/MS experiments were performed in product mode on a triple quadrupole mass spectrometer (Thermo Scientific TSQ Quantum Access Max).

Genome sequencing
The genomic DNA was extracted from the cultured bacterium according to Pospiech and Neuman [36] and the genome sequence was determined using a wholegenome shotgun (WGS) strategy with an Ion Torrent personal genome machine (Life Technologies). Quality filtered reads were in silico assembled via the DNASTAR NGen assembler by MR DNA (Shallowater, TX).
This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession LNUL00000000. The version described in this paper is LNUL01000000.

Genomic analysis
The L. coryniformis CRL 1001 genome sequence analysis was carried out following different approaches. Open Reading Frame (ORF) prediction and functional assignment was automatically performed using the RAST webserver (http://rast.nmpdr.org/), ORF Finder (http:// www.ncbi.nlm.nih.gov/gorf/gorf.html) and BLASTX (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The classification of the ORFs was carried out using BLASTP alignment against the database (http://blast.ncbi.nlm.nih.gov/ Blast.cgi), using a cut-off E-value of 1.10 −4 with at least 30 % of identity across a minimum of 50 % of a given protein length.

Comparative analyses
BLAST-based comparative analyses were performed of each one of the CDS of the cbi-cob-hem cluster between L. coryniformis CRL 1001 and two well-characterized Lactobacillus strains described as corrinoids compound producers with cobalamin activity, L. reuteri DSM 20016 and L. rossiae DSM 15814. This analysis was performed employing Easyfig 2.1: a genome comparison visualizer application [37].