Genome Insights into the Novel Species Microvirga brassicacearum, a Rapeseed Endophyte with Biotechnological Potential

Plants harbor a diversity of microorganisms constituting the plant microbiome. Many bioinoculants for agricultural crops have been isolated from plants. Nevertheless, plants are an underexplored niche for the isolation of microorganisms with other biotechnological applications. As a part of a collection of canola endophytes, we isolated strain CDVBN77T. Its genome sequence shows not only plant growth-promoting (PGP) mechanisms, but also genetic machinery to produce secondary metabolites, with potential applications in the pharmaceutical industry, and to synthesize hydrolytic enzymes, with potential applications in biomass degradation industries. Phylogenetic analysis of the 16S rRNA gene of strain CDVBN77T shows that it belongs to the genus Microvirga, its closest related species being M. aerophila DSM 21344T (97.64% similarity) and M. flavescens c27j1T (97.50% similarity). It contains ubiquinone 10 as the predominant quinone, C19:0 cycloω8c and summed feature 8 as the major fatty acids, and phosphatidylcholine and phosphatidylethanolamine as the most abundant polar lipids. Its genomic DNA G+C content is 62.3 (mol %). Based on phylogenetic, chemotaxonomic, and phenotypic analyses, we suggest the classification of strain CDVBN77T within a new species of the genus Microvirga and propose the name Microvirga brassicacearum sp. nov. (type strain CDVBN77T = CECT 9905T = LMG 31419T).


Introduction
After millions of years of evolution, microorganisms have compiled a noteworthy genetic diversity with biotechnological potential, which can be additionally enhanced by tools, such as genetic engineering or heterologous gene expression. Thus, microbial isolates are essential for diverse biotechnology-based industries, such as pharmaceutical biotechnology (where they play important roles in the production of antibiotics, antivirals, anticarcinogenic compounds, etc.), agricultural biotechnology (where they are applied as biocontrol agents or probiotics for plant growth promotion), and environmental biotechnology (with variate applications such as lignocellulose breakdown or degradation of xenobiotic compounds). Moreover, according to recent estimations, potentially 99.999% of microbial taxa remain undiscovered [1], so there is a huge genetic potential yet undiscovered.
In the last couple of decades, the extensive use of the PCR and sequencing of the 16S rRNA gene has allowed a more accurate bacterial identification and served as the base for the discovery of new bacterial colonies were regularly isolated to obtain pure cultures. Then, for long-term storage, isolated strains were stored in a sterile 20% glycerol solution at −80 • C.

DNA Extraction and 16S rRNA Amplification and Sequencing
CDVBN77 T DNA for amplifying and sequencing 16S rRNA gen was extracted using the REDExtract-n-AmpTM Plant PCR Kit (Sigma-Aldrich, Darmstadt, Germany) following the manufacturer's instructions. The amplification was performed using 2 µL of bacterial DNA (100 µg/mL) as the template, 6 µL of Milli-Q sterile water, 12.5 µL REDExtract Ready Mix, 2.5 µL 27F 1 µM (5 GAGGGTGGCGGTTCT 3 ), and 2,5 µL 1522R 1 µM (5 AAGGAGGTGATCCANCCRCA 3 ). The PCR conditions were the following: a first step of pre-heating at 95 • C for 9 min, 35 cycles of denaturing at 94 • C for 1 min; annealing at 56.5 • C for 1 min 30 s and extension 72 • C for 2 min, and a final extension at 72 • C for 7 min.

Draft Genome Sequencing and Annotation
Bacterial DNA for genome sequencing was obtained from cells grown in YMA medium at 28 • C for five days. DNA extraction was performed with the Quick DNA TM Fungal/Bacterial Miniprep Kit (Zymo Research®, Irvine, CA, USA) following the protocol provided by the manufacturer.
The draft genome sequence was obtained on an Illumina platform from the Genomics Service of the Institute of Functional Biology and Genomics (IBFG) (Salamanca, Spain). Sequences were assembled using Velvet 1.12.10, [25]. Gene calling, annotation, and search of genes related to plant growth promotion capabilities were performed using RAST Pipeline [26]; the BlastKOALA tool was also used to perform KEGG Orthology (KO) assignment based on the KEGG database [27] in order to complete the mining of genes related to plant growth promotion; the dbCAN2 meta server [28] was used to identify the coding sequences (CDSs) encoding carbohydrate-active enzymes (CAZymes) using HMMER annotation; and the analysis of gene clusters related to secondary metabolites production was performed using antiSMASH 5.0 webserver [29]. This genome sequence was deposited at the DDBJ/EMBL/GenBank under the accession VCMV00000000. The version described in this paper is version VCMV01000000.

Genotypic Analysis of Microvirga brassicacearum CDVBN77 T
The GenBank accession number of the CDVBN77 T 16S rRNA gene sequence is MK938302. Phylogenetic analysis of the CDVBN77 T 16S rRNA gene sequence and the sequences of the type strains of all the species included within the genus Microvirga was performed using MEGA7 software [30], based on the alignment of the sequences with Clustal_W [31]. The distances were calculated using Kimura's two-parameter model [32], and the phylogenetic tree was inferred using neighbor-joining (NJ; [33]) analysis. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the draft genome sequence of strain CDVBN77 T and those of its closest related species M. aerophila c27j1 T (GenBank/EMBL/DDBJ accession number NZ_QOIO00000000.1) and M. flavescens DSM 21344 T (GenBank/EMBL/DDBJ accession number QMKH00000000) were calculated using the ANI calculator (www.ezbiocloud.net/tools/ani) and the Genome-to-Genome Distance Calculator (GGDC 2.1) (http://ggdc.dsmz.de/distcalc2.php), respectively. The mol % G+C content of DNA was determined from the draft genome sequence.

Chemotaxonomic Analysis of Microvirga brassicacearum CDVBN77 T
For the analysis of fatty acid methyl esters (FAMEs), biomass was harvested as detailed by Weon et al. [8]. For the analyses of polar lipids and respiratory quinones, biomass was harvested after cultivation of the bacterium for seven days in R2A broth medium (dextrose (0.5 g/L), dipotassium phosphate (0.3 g/L), casamino acids (0.5 g/L), magnesium sulphate (0.05 g/L), proteose peptone (0.5 g/L), sodium pyruvate (0.3 g/L), starch (0.5 g/L), and yeast extract (0.5 g/L)) at 28 • C and 150 rpm shaking. Analysis of FAMEs was performed as previously described [34]. For the identification of polar lipids and respiratory quinones, cells collected from the plates into sterile Falcon®tubes were freeze dried and sent to the Identification Service of the German Collection of Microorganisms and Cell Cultures DSMZ.
Cell morphology and flagella were observed under a Zeiss EM209 transmission electron microscope, after growth in YMA medium at 28 • C for five days and cell treatment with 2% uranyl. To assay salt tolerance, the bacterium was cultivated in YMB medium (mannitol (7.0 g/L), yeast extract (2 g/L), dipotassium phosphate (0.2g/L), and magnesium sulphate (0.2g/L)) supplemented with 0, 0.5, 1, 1.5, and 2% (w/v) NaCl. The same medium adjusted to a final pH range of 3-11 was used to examine the growth capability at different pHs; in both cases, flasks containing 50 mL of the different media were inoculated with a 10 µl sterile plastic loop containing cells collected from a two-day plate culture of the bacterium. After the inoculation, flasks were cultivated in a shaker at 150 rpm for two days at 28 • C. Plates containing YMA medium (YMB plus 2% agar) were incubated at 4, 12, 25 and 37 • C to determine the temperature range for optimal growth. In all cases, the cultures were examined at 2, 5, 10, and 15 days after inoculation.
Enzymatic assays were performed using the p-nitrophenyl (pNP) substrates listed in Table 1. Substrates were used at a concentration of 0.2% in 50 mM phosphate buffer (pH 7); however, those requiring pH 8.5 were prepared in Tris-HCl 0.2 M buffer and those requiring pH 5.0 were prepared in an acetate buffer (0.2 M). The enzymatic reactions were carried out in a multi-well plate mixing 50 µL of substrate prepared as specified above and 50 µL of a suspension of bacteria in sterile water with 6 × 10 9 CFU/mL obtained from cells previously grown in YMA medium for 15 days at 28 • C. Plates with the reaction mixture were incubated at 28 • C for 24 h and then revealed with 100 µL per well of a 4% sodium carbonate solution. A positive reaction was evidenced by the appearance of a yellow color within a few seconds.
To evaluate siderophore production, strain CDVBN77 T was inoculated in M9-CAS-agar medium plates [37] with the modifications suggested by Alexander and Zuberer [38] after 21 days of growth at 28 • C. The addition of hexadecyltrimethylammonium bromide (HDTMA) as a cationic solvent was used to stabilize the Fe-CAS complex, which provides the characteristic orange-colored halo around the colonies when siderophores are produced.
The solubilization of non-soluble phosphates into soluble assimilable ions was analyzed in Pikovskaya medium plates [39], which contain calcium phosphate (Ca 2 HPO 4 ) as P source. The presence of clear halos surrounding bacterial colonies are indicative of phosphate solubilization. Strain CDVBN77 T was inoculated in Pikovskaya medium and phosphate solubilization was evaluated after 21 days of growth at 28 • C.
Cellulose production was determined as described by Robledo et al. [40] in Nutrient Agar (NA) plates containing Congo Red colorant, which binds to cellulose polymers. Red-colored colonies are indicative of cellulose production. Strain CDVBN77 T was inoculated in plates with Congo Red and the color of the colonies was evaluated after 21 days of growth at 28 • C.
Other phenotypic characters were tested by using the API 20NE and API ZYM (bioMerieux ® ) miniaturized galleries following the manufacturer's instructions.

Bacterial Isolation and Identification
Strain CDVBN77 T was isolated as part of a collection of rapeseed root endophytes from plants sampled at the municipality of Castellanos de Villiquera (province of Salamanca, Spain) [22]. The comparison of the 16S rRNA gene sequence with those of type strains of described species available in GenBank and EzBiocloud databases showed that all strains but CDVBN77 T showed similarities higher than 98% with their closest related type strains, whereas the similarity of strain CDVBN77 T with M. aerophila DSM 21344 T , its closest related type strain, was only 97.64%, which according to the threshold value of 98.7% in the 16S rRNA gene sequence with the closest related species indicated by Jongsik and collaborators [2] indicates that the strain constitutes a new species within the genus Microvirga.
Therefore, we performed a phylogenetic analysis of the 16S rRNA gene sequence of strain CDVBN77 T and those of the type strains of all species included within the genus Microvirga.
Phylogenetic trees constructed with both Maximum Likelihood (ML) and Neighbor Joining (NJ) methods showed a similar topology ( Figure 1) with strain CDVBN77 T appearing in a separated branch with M. flavescens c27j1 T and M. aerophila DSM 21344 T as the closest related species. Thus, the phylogenetic analysis of the 16S rRNA gene supports the classification of the bacterium CDVBN77 T as a new species within the genus Microvirga.

Genome Properties and Comparison with Those of Its Closest Related Species
The genome sequence of strain CDVBN77 T was obtained in order to be compared with those of its closest related species. Genome properties of the strain based on RAST annotations are detailed in Table 2.

Genome Properties and Comparison with Those of Its Closest Related Species
The genome sequence of strain CDVBN77 T was obtained in order to be compared with those of its closest related species. Genome properties of the strain based on RAST annotations are detailed in Table 2.
ANI values between the genome sequence of CDVBN77 T and M. flavenscens c27j1 T and M. aerophila DSM 21344 T were 76.14% and 77.23%, respectively. Considering the recommended ANI threshold value for species delimitation of 95% to 96% [41], these genome comparisons clearly support the classification of strain CDVBN77 T as a novel species of the genus Microvirga.
The results of dDDH between the genome sequence of CDVBN77 T and M. flavescens c27j1 T and M. aerophila DSM 21344 T were 20.50% and 21.20%, respectively. Since the dDDH threshold value for species delineation has been established at 70% [42], we can conclude that the bacterial isolate CDVBN77 T does not belong to its closest related species and should be classified within a new species of the genus Microvirga.

Plant Growth Promotion Potential
The genome mining revealed many genes involved in PGP mechanisms. Regarding the acquisition of nutrients, we found some mechanisms involved in the solubilization of P and K, making their uptake easier for the plant. The genome annotations showed genes encoding for proteins with phosphatase activity, one of the main mechanisms for P solubilization, such as the enzymes alkaline phosphatase (EC 3.1.3.1), exopolyphosphatase (EC 3.6.1.11), inorganic triphosphatase (EC 3.6.1.25) and pyrophosphatase (EC 3.6.1.1), polyphosphate kinase (EC 2.7.4.1), and a pyrophosphate-energized proton pump (EC 3.6.1.1) [43]. The glucose dehydrogenase PQQ-dependent enzyme (EC 1.1.5.2), which is responsible of the oxidation of glucose into the phosphate solubilizing molecule gluconic acid [44,45], was also found in this search. In addition, some other enzymes involved in the production of P and K solubilization acids are encoded by this bacterium, such as citrate synthase (EC 2.3.3.1) and malate synthase G (EC 2.3.3.9) [46]. Moreover, regarding the transport of these two elements, the genome encodes transport systems such as the Pst system for P [47] or the Kup and Kef systems for K [48]. Regarding the iron supply, we found some genes annotated as siderophores or other iron-associated transport proteins, such as the ferrichrome-iron receptor, a ferric iron ABC transporter, a ferric hydroxamate ABC transporter (TC 3.A.1.14.3), the ATP-binding protein FhuC, as well as proteins of the Fec system, the AfuABC system (Iron (III)), and the SitA system (Iron(II)/manganese). Finally, the capability to fix atmospheric nitrogen into a plant's available state of nitrogen [49] is a common mechanism in rhizobial strains. In this case, we found the presence of genes implicated on Fix and Nif systems.
A prior step to endophytic living is to attach and form biofilms over the root surface. Bacteria commonly produce exopolysaccharides in order to colonize the root surface [50]. Genome annotations revealed a great number of genes related to the synthesis or transport of exopolysaccharides, for example, the exoD gene, which is associated with this process [51], lpx genes, responsible for the synthesis of this type of molecules [52], or lpt [53] and Rf [54] transport systems. Once inside the plant, the bacterium must evade the plant defense. The genome annotations revealed that the bacterium encodes genes for the production of salicylate 1-hydroxylase (EC 1.14.13.1), an enzyme that degrades the salicylic acid into catechol.
Moreover, strain CDVBN77 T grew on chrome azurol S (CAS) indicator medium, where the colonies were surrounded by a yellow-orange halo (2 mm radius around colonies), indicating siderophore production.
In addition, CDVBN77 T solubilized phosphate on Pikovskaya's agar, forming halos around its colonies (2 mm radius around colonies). Additionally, the results obtained using pNP-based substrates showed the production of phosphatases (with activity at acid, alkaline, and neutral pH) and bisphosphatases (with activity at alkaline pH).
Furthermore, strain CDVBN77 T colonies were red after seven days of incubation on plates containing Congo Red. The intensity of the color was comparable to that recorded for other strains, which indicated that CDVBN77 T produces a moderate amount of a polysaccharide with 1,4-β-glycosidic bonds.

Potential for the Production of Enzymes with Biotechnological Potential
Analysis of the genome sequence of strain M. brassicacearum CDVBN77 T with dbCAN2 showed 115 genes encoding different CAZymes involved in degradation, modification, or creation of glycosidic bonds.
In summary, we identified gene modules belonging to five different enzyme classes: (i) glycoside hydrolases (GHs), enzymes that catalyze the hydrolysis of glycosidic linkage of glucoside-30 gene counts in 15 different families; (ii) glycosyltransferases (GTs), involved in the formation of glycosidic bonds-46 gene counts in 11 different families; (iii) polysaccharide lyases (PLs), which perform non-hydrolytic cleavage of glycosidic bonds-2 gene counts in 2 different families; (iv) carbohydrate esterases (CEs), which hydrolyze carbohydrate esters-22 gene counts in 6 different families; and (v) auxiliary activities (AAs), redox enzymes that act in conjunction with CAZymes-15 gene counts in 5 different families (Table 3).    Moreover, enzymatic assays based on pNP-based substrates show the capability of the strain to synthesize β-galactosidase, β-glucosidase, and α-rhamnosidase. Nevertheless, in the conditions assayed, we could not detect the production of xylosidases or arabinofuranosidases.

Genome Mining of Gene Clusters Associated to the Biosynthesis of Secondary Metabolites
AntiSMASH output revealed five biosynthetic gene clusters (BGCs) involved in the secondary metabolism of the bacterium.
One of those clusters encodes an undescribed terpene BGC in which the core gene is a phytoene synthase (EC 2.5.1.32), an enzyme involved in the first steps of the biosynthesis of carotenoids. According to antiSMASH clusterblast, this cluster seems to be distributed among different Ochrobactrum and Brucella genomes, but it does not appear in the seven genomes of strains belonging to the Microvirga genus included in the database at the moment of writing this work.
Another BGC found in Microvirga brassicacearum CDVBN77 T is related to the synthesis of a N-acyl-homoserine lactone. This family of molecules is often involved in bacterial quorum sensing [55]. In this case, the cluster is present in other Microvirga genomes, but it has never been tested for the production of any specific molecule.
Microvirga brassicacearum CDVBN77 T also encodes two nonribosomal peptide synthetases (NRPSs) BGCs that are neither described for the production of an already known molecule. Moreover, none of them share similitude with any region of any other Microvirga strains' genomes, although it appears related to sequences of strains belonging to Pseudomonas and Chelatococcus genera (with 6%-20% genes showing similarity).
In addition, a type III polyketide synthase (T3PKS) is present, a type of BGC involved in the synthesis of a great diversity of molecules derived from the metabolism of fatty acids [56]. The region encoding this BGC shares certain similitude with regions of some Pseudomonas genomes but not in the core genes, and none of them has been described for the production of a known metabolite.

Colony and Cellular Morphology in Strain CDVBN77 T
After seven days of incubation at 28 • C in R2A agar medium, strain CDVBN77 T forms circular, flat, and transparent colonies with entire borders. The cells are rod-shaped, 1.2-1.5 µm in length and 0.7-0.9 µm in width, and motile by means of a polar flagellum (Figure 2).

Phenotypic and Chemotaxonomic Characterization of Strain CDVBN77 T
The analysis of respiratory quinones revealed that ubiquinone-10 (Q-10) is the major quinone in CDVBN77 T , and the main polar lipids of this strain are supporting the classification of the bacterium within the genus Microvirga [11].
The major fatty acids in strain CDVBN77 T are C 19:0 cyclo ω8c (24.3%) and Summed Feature 8 (39.3%) ( Table 4), as it occurs for its closest related strains M. flavescens c27j1 T [17] and M. aerophila DSM 21344 T [10], as well as for the type species of the genus M. subterranea DSM 14364 T [11], supporting the classification of the strain within the genus Microvirga. However, the fatty acid profile of this strain significantly differs from that of the closest related species (Table 4), supporting the classification of CDVBN77 T into a different species within the genus. Table 4. Cellular fatty acid composition (%) of Microvirga brassicacearum CDVBN77 T (data from this study) and its closest related species M. flavescens c27j1 T (data from Zhang et al. [17]) and M. aerophila DSM 21344 T (data from Weon et al. [10]), as well as for the type species of the genus M. subterranea DSM 14364 T (data from Kanso and Patel [11] Phenotypic features analyzed in strain CDVBN77 T are detailed in the species protologue, and the main phenotypic differences with the closest related species and the type species of the genus are summarized in Table 5. Table 5. Phenotypic differences between Microvirga brassicacearum CDVBN77 T (data from this study) and its closest related species M. flavescens c27j1 T (data from Zhang et al. [17]) and M. aerophila DSM 21344 T (data from Weon et al. [10]), as well as for the type species of the genus M. subterranea DSM 14364T (data from Kanso and Patel [11] Gelatin

Discussion
Many plant endophytic strains have been described as new taxa [57][58][59][60]. In this study, we isolated strain CDVBN77 T , whose 16S rRNA gene sequence showed a relatively low similarity with its closest related species, M. aerophila DSM 21344 T (97.64%) and M. flavescens c27j1 T (97.50%), which suggested that it could belong to a new species within the genus Microvirga. Several Microvirga species, namely, M. lupini, M. lotononidis, M. zambiensis, M. ossetica, and M. vignae [18][19][20], were for the first time isolated as legume endophytes. The phylogenetic analysis of this strain based on the 16S rRNA gene showed that the bacterium appears in a separated branch within the genus Microvirga but clustered with its two closest related type strains M. flavescens c27j1 T and M. aerophila DSM 21344 T . The presence of Q-10 as the major quinone and C19:0 cyclo ω8c and Summed Feature 8 as the main fatty acids supports its classification within the genus Microvirga. However, this isolate differs from the closest species, M. aerophila and M. flavescens, in the capability to hydrolyze gelatin and aesculin, positive for M. brassicacearum CDVBN77 T and negative for the other two, and the color of the colonies in R2A agar medium, transparent in M. brassicacearum CDVBN77 T and light yellow and pale pink in M. flavescens and M. aerophila, respectively. Unlike M. aerophila, it reduces nitrates to nitrites and assimilates D-glucose and L-arabinose, and it does not produce the enzyme β-glucoronidase; moreover, in contrast to M. flavescens, it produces trypsin. Thus, the results of the phylogenetic, chemotaxonomic, and phenotypic analysis clearly support the classification of strain CDVBN77 T as a new species within the genus Microvirga, for which the name Microvirga brassicacearum sp. nov. is proposed.
The genus Microvirga belongs to Rhizobiales, a broadly studied order in the field of plant-bacteria interactions because of its role as plant growth promoter. Five species of the genus Microvirga have been described as plant endosymbionts capable of promoting plant growth, namely, M. lupini, M. lotononidis, M. zambiensis [18], M. vignae [20], and M. ossetica [19]. The genome mining of strain M. brassicacearum CDVBN77 T shows the presence of genes implicated in the nutrient provision to the plant, such as P and Fe, capabilities also proven in in vitro tests, as well as K and N. Moreover, both the analysis of the genome sequence as well as in vitro tests show the capability of this strain to synthesize exopolysaccharides which may allow the attachment of the bacterial cells to the plant root surface in order to establish an interaction with the host.
To live as plant endophytes, bacteria must have genetic machinery to use plant-synthesized compounds as a source of nutrients. Some of the enzymes implicated in plant compound degradations are very interesting for industries based on biomass degradation, such as bioethanol production, pulp and paper industries, beverages industries, medicinal and analytical chemistry, fabrication of detergents, and textile de-sizing [6]. Thus, we explored the enzymatic machinery related to carbohydrate degradation encoded by the M. brassicacearum CDVBN77 T genome. As a result, we identified genes related to the degradation and modification of biomass polymers, such as cellulose and hemicelluloses. β-glucosidase is the key enzyme in the cellulase system [34,61,62], since it converts cellobiose to glucose, completing the final step during cellulose hydrolysis. In the genome of M. brassicacearum CDVBN77 T , we have found five genes belonging to family GH1 and three genes belonging to family GH3; both enzyme families include enzymes, such as β-glucosidases (EC 3.2.1.21) or even exoglucanases (EC 3.2.1.74), enzymes which are also involved in the cellulose polymer degradation [63].
The complete degradation of hemicelluloses requires the combined activity of xylanases, β-xylosidases, and several accessory enzymes such as α-arabinofuranosidases [64]. In the genome of Microvirga brassicacearum CDVBN77 T , we have found gene counts in the GH1, GH2, and GH3 families, all of them including enzymes with xylan 1,4-β-xylosidase (EC 3.2.1.37) activity. Moreover, GH2 and G3 families include enzymes with α-arabinofuranosidase activity. However, we could not detect the in vitro synthesis of xylosidases and arabinofuranosidases, either because the annotated genes belonging to the GH1, GH2, and GH3 families have other different activities, or because they were not induced under the assayed conditions.
On the other hand, we found how this bacterium is able to hydrolyze several substrates linked to p-nitrophenol, which reveals that the bacterium synthesizes α-rhamnosidase, with potential applications in the clarification and debittering of citrus juices, the enhancement of wine aromas, or the synthesis of certain pharmaceutical compounds [65] and β-galactosidase, with potential application in food processing industries for the hydrolysis of lactose in milk and milk by-products [66].
Regarding the secondary metabolism prospection of the bacterium, we found an interesting niche for researching secondary metabolite BGCs that have not been very studied within the Microvirga genus. AntiSMASH results showed that the CDVBN77 T strain has the potential to encode at least five undescribed secondary metabolites; amongst those, a terpene, a PKS, and two NRPS have potential applications in industry. NRPS are widely distributed among many taxa, and their products, nonribosomal peptides, are characterized because of their broad range of biological activities, such as being mainly antimicrobials, but also antitumoral compounds, antivirals, siderophores, etc. [67][68][69][70]. The same fact happens with type III polyketides, in which we also found interesting activities [54,71,72]. In addition, carotenoids (terpenes) are also used in the pharmaceutical and food industries because of their antioxidant activities or colorant properties [73,74].
Thus, in this study we found an interesting genetic potential in the genome of M. brassicacearum CDVBN77 T to synthesize enzymes and secondary metabolites. Moreover, we detected in vitro the production of some of the predicted enzymes and metabolites. Nevertheless, the purification of such gene products and the determination of their specific mechanisms of action in addition to the conditions of pH and temperature in which the molecules are active are required in order to determine the possible industrial interest of those substances.

Description of Microvirga brassicacearum sp. nov.
Microvirga brassicacearum (bras.si.ca.ce.a.rum. M.L. fem. pl. gen. n. brassicacearum of the Brassicaceae, referring to its isolation from the rhizoplane of plants belonging to the genus Brassica). Cells are rod-shaped, 1.2-1.5 µm in length and 0.7-0.9 µm in width, and motile by means of a polar flagellum. In 869(1/10) medium, colonies are transparent, circular, and flat, with an average size of 0.1-0.4 mm in diameter after seven days of growth at 28 • C in R2A medium. The temperature growth ranges between 12 and 37 • C, with an optimum of 28 • C. The growth at different pH ranges occurs between 6 and 10 with an optimum of 7. It grows between 0 and 1.5% NaCl in 869(1/10) liquid medium. Catalase and oxidase positive. The main polar lipids are phosphatidylcholine and phosphatidylethanolamine. Ubiquinone-10 (Q-10) is the major respiratory quinone and C19:0 cyclo ω8c and Summed Feature 8 are the main fatty acids. Results obtained in the API 20NE system indicate that the strain hydrolyzes aesculin, but it does not ferment D-glucose and does not hydrolyze arginine, urea, and gelatin. Reduction of nitrates to nitrites is positive. The assimilation of D-glucose, L-arabinose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, malic acid, and trisodium citrate is positive and that of D-mannose, capric acid, adipic acid, and phenylacetic acid is negative. Results obtained in the API ZYM system showed activity in the enzymes alkaline phosphatase, esterase (C 4), esterase lipase (C 8), leucine arylamidase, trypsin, acid phosphatase, and naphtol-AS-BI-phosphohydrolase, but activity was not shown in lipase (C14), valine arylamidase, cysteine arylamidase, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, β-glucosidase, α-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase.
The G+C base composition was 62.3 mol %. The type strain, CDVBN77 T (= LMG 31419 T = CECT 9905 T ), was isolated as an endophyte from Brassica napus roots in Castellanos de Villiquera (Spain).