Biotechnological and Ecological Potential of Micromonospora provocatoris sp. nov., a Gifted Strain Isolated from the Challenger Deep of the Mariana Trench

A Micromonospora strain, isolate MT25T, was recovered from a sediment collected from the Challenger Deep of the Mariana Trench using a selective isolation procedure. The isolate produced two major metabolites, n-acetylglutaminyl glutamine amide and desferrioxamine B, the chemical structures of which were determined using 1D and 2D-NMR, including 1H-15N HSQC and 1H-15N HMBC 2D-NMR, as well as high resolution MS. A whole genome sequence of the strain showed the presence of ten natural product-biosynthetic gene clusters, including one responsible for the biosynthesis of desferrioxamine B. Whilst 16S rRNA gene sequence analyses showed that the isolate was most closely related to the type strain of Micromonospora chalcea, a whole genome sequence analysis revealed it to be most closely related to Micromonospora tulbaghiae 45142T. The two strains were distinguished using a combination of genomic and phenotypic features. Based on these data, it is proposed that strain MT25T (NCIMB 15245T, TISTR 2834T) be classified as Micromonospora provocatoris sp. nov. Analysis of the genome sequence of strain MT25T (genome size 6.1 Mbp) revealed genes predicted to responsible for its adaptation to extreme environmental conditions that prevail in deep-sea sediments.


Introduction
Novel filamentous actinobacteria isolated from marine sediments are a prolific source of new specialized metabolites [1][2][3], as examplified by the discovery of the abyssomicins, a new family of polyketides [4] produced by Micromonospora (formerly Verrucosispora) maris [5] and the proximicins, novel aminofuran antibiotics and anticancer compounds isolated from Micromonospora (Verrucosispora) fiedleri [6,7]. Novel micromonosporae have 2.1. Isolation, Maintenance and Characterization of Strain MT25 T Micromonospora strain MT25 T was isolated from a sediment sample (no. 281) taken from the Mariana Trench (Challenger Deep; 142 • 12 372 E; 11 • 19 911 N) using a standard dilution plate procedure [19] and raffinose-histidine agar as the selective medium [20]. The sediment was collected at a depth of 10,898 m by the remotely operated submersible Kaiko, using a sterilized mud sampler during dive 74 [21]. The sample (approximately 2 mL) was taken to the UK in an isolated container at 4 • C, then stored at −20 • C.

Compound Identification
Compound 1 was obtained as a white amorphous powder, 16
Seven hydrogen resonances lacked correlations in the 1 H-13 C HSQC spectrum of 2 and were therefore recognized as being located on either oxygen or nitrogen. From the results of a 1 H-15 N HSQC measurement made with 2 it was evident that four protons were With all protons assigned to their directly bonded carbon and nitrogen atoms it was possible to deduce substructures with the aid of the 1 H-1 H COSY spectrum of 1 ( Figure 3). The connectivities between substructures were established from key 1 H-13 C HMBC correlations ( Figure 3). Thus, correlations between C-2 (δ C 173.8) and H 2 -3, H 2 -1 and between C-11 (δ C 173.8) and H 2 -10, H 2 -12 as well as between C-5 (δ C 52.7) and H 2 -3 and between C-8 (δ C 52.1) and H 2 -17 and between C-14 (δ C 169.7) and H-5, H-13, H 3 -15 and between C-16 and H-8, H 2 -9 and H 2 -17 clearly defined the planar structure as shown in 1. Finally, the positions of nitrogen were defined from 1 H-15 N-HMBC which showed long range correlations between N-13 and H4a/b and H3-15 and between N-7 and H9a/b ( Figure 3 and Figure S9). Given these results and comparisons with previously data [22], the compound was identified as n-acetylglutaminyl glutamine amide.
Compound 2 was identified as deferoxamine B. Its molecular formula was established as C 25 (Table 1), and by comparing it with the previously reported data on desferrioxamine [23].
Seven hydrogen resonances lacked correlations in the 1 H-13 C HSQC spectrum of 2 and were therefore recognized as being located on either oxygen or nitrogen. From the results of a 1 H-15 N HSQC measurement made with 2 it was evident that four protons were bonded to nitrogen: comprising one NH 2 group; NH 2 -1 and two NH groups; NH-12 [δ With all protons assigned to their directly bonded carbon and nitrogen atoms it was possible to deduce substructures. The connectivities between these substructures were established from key 1 H-13 C HMBC and 1 H-15 N HMBC correlations ( Figure 3 and Figure S21). The positions of nitrogen in amide formation were confirmed by 15 Figure S21). The 1,1-ADEQUATE experiment confirmed the correlations of 1 H-1 H COSY and partial substructures through its two bond correlations ( Figure 3 and Figure S23). The 1,1-ADEQUATE is a technique used to obtain heteronuclear correlations similarly to 1 H-13 C HMBC. While correlation signals from HMBC do not separate 2 J CH from 3 J CH , 1,1-adequate, which exclusively observes 1 J CH and 2 J CH , and can be combined with 1 H- 13

Genome Sequencing and Annotation
The whole genome sequencing reads of strain MT25 T , generated using an Ion Torrent PGM instrument, 316v2 chips and Ian on PGM Hi-QTM View Sequencing Kit, were assembled using the Ion Torrent SPAdes plugin (v. 5.0.0.0) program (Life Technologies Limited, Paisley, UK). The size of whole genome sequence of the strain represented by 1170 contigs is 6,053,796 bp with a G + C content of 71.6%. Additional genomic features of the strain are shown in Table 2 according to GenBank NCBI prokaryotic genome annotation pipeline [24][25][26].

Phylogeny
The phylogenetic tree ( Figure 4) based on almost complete 16S rRNA gene sequences shows that Micromonospora strain MT25 T belongs to a well-supported lineage together with the type strains of nine Micromonospora species. It is most closely related to M. chalcea DSM 43026 T . With only 4 nucleotides difference within a 1437 sequence, the 16S rRNA sequences of these two strains are 99.7% identical. The 16S rRNA of strain MT25 T also shares a relatively high sequence identify with the Micromonospora aurantiaca [27], Micromonospora marina [28], Micromonospora maritima [29], Micromonospora sediminicola [30] and Micromonospora tulbaghiae [31,32] strains. The close relationship between these species is in a good agreement with the results from previous 16S rRNA gene sequence analyses [8,33]. The sequence similarities between the 16S rRNA sequences of strain MT25 T and the other Micromonospora strains range from 88.6 to 99.1%, which is equivalent to 13 to 20 nucleotide differences.
shows that Micromonospora strain MT25 T belongs to a well-supported lineage together with the type strains of nine Micromonospora species. It is most closely related to M. chalcea DSM 43026 T . With only 4 nucleotides difference within a 1437 sequence, the 16S rRNA sequences of these two strains are 99.7% identical. The 16S rRNA of strain MT25 T also shares a relatively high sequence identify with the Micromonospora aurantiaca [27], Micromonospora marina [28], Micromonospora maritima [29], Micromonospora sediminicola [30] and Micromonospora tulbaghiae [31,32] strains. The close relationship between these species is in a good agreement with the results from previous 16S rRNA gene sequence analyses [8,33]. The sequence similarities between the 16S rRNA sequences of strain MT25 T and the other Micromonospora strains range from 88.6 to 99.1%, which is equivalent to 13 to 20 nucleotide differences. Greater confidence can be placed in the topology of phylogenetic trees based on whole genome sequences than on corresponding 16S rRNA gene trees, as the former are generated from millions, as opposed to hundreds, of unit characters [5]. The phylogenomic tree ( Figure 5) shows that the strain MT25 T is most closely related to M. tulbaghiae DSM 45124 T . In turn, these strains belong to a well-supported lineage which includes the M. aurantiaca, M. chalcea, M. marina, M. maritima and M. sediminicola strains together with the type strain of M. humi [34], all of these species belong to a distinct taxon, group 1a, highlighted in the genome-based classification of the genus Micromonospora generated by Carro et al. [8]. Greater confidence can be placed in the topology of phylogenetic trees based on whole genome sequences than on corresponding 16S rRNA gene trees, as the former are generated from millions, as opposed to hundreds, of unit characters [5]. The phylogenomic tree ( Figure 5) shows that the strain MT25 T is most closely related to M. tulbaghiae DSM 45124 T . In turn, these strains belong to a well-supported lineage which includes the M. aurantiaca, M. chalcea, M. marina, M. maritima and M. sediminicola strains together with the type strain of M. humi [34], all of these species belong to a distinct taxon, group 1a, highlighted in the genome-based classification of the genus Micromonospora generated by Carro et al. [8].
The recommended thresholds used to distinguish between closely related prokaryotic species based on average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values are 95 to 96% [35,36] and 70% [36,37], respectively. Table 3 shows that the ANI and dDDH similarities between strain MT25 T and M. aurantiaca ATCC 27029 T , M. chalcea DSM 43026 T and M. marina DSM 45555 T , its three closest phylogenomic neighbors, are below the cut-off points used to assign closely related strains to the same species. The ANI and dDDH values also provide further evidence that strain MT25 T is most closely related to M. tulbaghiae DSM 45142 T . However, the relationship between these strains is not clear-cut as they share a dDDH value below the 70% threshold and an ANI value at the borderline used to assign closely related strains to the same species. Conflicting results such as these are not unusual, as exemplified by studies on closely related Micromonospora and Rhodococcus species [33,38]. In such instances, ANI and dDDH similarities need to be interpreted with a level of flexibility and should also be seen within the context of other biological features, such ecological, genomic and phenotypic criteria [33,38,39]. Again, the use of a universal ANI threshold for the delineation of prokaryotic species has been questioned [40].  The recommended thresholds used to distinguish between closely related prokaryotic species based on average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values are 95 to 96% [35,36] and 70% [36,37], respectively. Table 3 shows that the ANI and dDDH similarities between strain MT25 T and M. aurantiaca ATCC 27029 T , M. chalcea DSM 43026 T and M. marina DSM 45555 T , its three closest phylogenomic neighbors, are below the cut-off points used to assign closely related strains to the same species. The ANI and dDDH values also provide further evidence that strain MT25 T is most closely related to M. tulbaghiae DSM 45142 T . However, the relationship between these strains is not clear-cut as they share a dDDH value below the 70% threshold and an ANI value at the borderline used to assign closely related strains to the same species. Conflicting results such as these are not unusual, as exemplified by studies on closely related Micromonospora and Rhodococcus species [33,38]. In such instances, ANI and dDDH similarities need to be interpreted with a level of flexibility and should also be seen within the context of other biological features, such ecological, genomic and phenotypic criteria [33,38,39]. Again, the use of a universal ANI threshold for the delineation of prokaryotic species has been questioned [40].  The tree is rooted using the type strain of Catellatospora citrea.

Species Assignment
It can be seen from Table 4 that strain MT25 T and M. tulbaghiae DSM 45142 T , its closest phylogenomic neighbor, have phenotypic features in common though a range of other properties can be weighted to distinguish between them. Strain MT25 T , unlike the M. tulbaghiae strain, grows at pH 6 and 10, reduces nitrate and shows much greater activity in the AP1-ZYM tests. In contrast, the M. tulbaghiae strain, unlike strain MT25 T , grows at 4 • C, in the presence of 5% w/v sodium chloride, produces hydrogen sulfide and shows greater activity in the degradation tests. In addition, strain MT25 T produces sessile, rugose ornamental single spores on the substrate mycelium (Figure 1) whereas the M. tulbaghiae strain bears smooth, single spores borne on sporophores [31]. Further, strain MT25 T produces an orange as opposed to a brown substrate mycelium on yeast-malt extract agar though the colonies of both strains become dark brown/black on sporulation. The two strains also have different cellular sugar profiles as only strain MT25 T produces mannose. They can also be distinguished using a range of genomic features, notably genome size and G + C content. The genome size of strain MT25 T is 6.05 Mbp and its G + C content is 71.6%, whilst the corresponding figures for the M. tulbaghiae strain are 6.5 Mbp and 73.0%. Genome size and G + C content are considered to be conserved within species and can therefore represent useful taxonomic markers [5]. Inter-species variation in genomic G + C content does not usually exceed 1% [5,41].

Tolerance tests:
Growth at 4 • C -+ Growth at pH 6.0 and pH 10 + -Growth in presence of 5% w/v NaCl -+
In light of all of these data, it can be concluded that although strains MT25 T and M. tulbaghiae DSM 45142 T are close phylogenomic neighbors which can be distinguished using a combination of genomic and phenotypic properties, notably their genome sizes and G+C contents. It is, therefore, proposed that isolate MT25 T be considered as the type strain of a novel Micromonospora species that belongs to the phylogenomic group 1a, as designed by Carro et al. [8]. The name proposed for this species is Micromonospora provocatoris sp. nov.

Description of Micromonospora provocatoris sp. nov.
Micromonospora provocatoris (pro.vo.ca.to'ris. L. gen. n. provocatoris, of a challenger, referring to the Challenger Deep of the Mariana Trench, the source of the isolate), Aerobic, Gram-positive strain, non-acid-fast actinobacterium which forms nonmotile, single, sessile spores (0.8-0.9 µm) with rugose ornamentation on extensively branched substrate hyphae, but does not produce aerial hyphae. Colonies are orange on oatmeal agar eventually turning black on sporulation ( Figure S2). Growth Occurs between pH 6.0 and 8.0, optimally at pH 7.0, from 10 • C to 37 • C, optimally at 28 • C and in the presence of 1% w/v sodium chloride. Aesculin is hydrolyzed and catalase produced. Degrades arbutin and L-tyrosine, but not starch or xylan. Furthermore, acid and alkaline phosphatases, α-chymotrypsin, cystine, leucine and valine arylamidases, esterase (C4), lipase esterase (C8), lipase (C14), β-galactosidase, β-glucosidase, naphthol-AS-BI-phosphohydrolase and trypsin are produced, but not α-fucosidase, α-galacturonidase, β-glucuronidase or α-mannosidase. The cell wall contains meso-A 2 pm, and the whole cell sugars are glucose, mannose, ribose and xylose. The predominant fatty acid is iso-C16:0 and the polar lipid profile contains diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol, a glycolipid and two unidentified phospholipids. The dDNA G + C content of the type and only strain is 71.6% and it is genome size 6.05 Mbp.
The type strain MT25 T (= NCIMB 15245 T = TISTR 2834 T ) was isolated from surface sediment from the Challenger Deep in the Mariana Trench of the Pacific Ocean. The accession numbers of the 16S rRNA gene sequence and that of the whole genome of the strain are AY894337 and QNTW00000000, respectively.

Specialised Metabolite-Biosynthetic Gene Clusters
Antibiotic and Secondary Metabolites Analysis Shell "AntiSMASH 6.0.0 0 alpha 1" [42] predicts natural products-biosynthetic gene clusters (NP-BGCs) that are based on the percentage of genes from the closest known bioclusters which share BLAST hits to the genome of the strains under consideration. Mining the draft genome of M. provocatoris MT25 T revealed the presence of ten known BGCs (Table 5). Two gene clusters were predicted to be responsible for the biosynthesis of siderophore desferrioxamine B, which was initially isolated from Streptomyces strain 1D38640 [43], and rhizomide A, which has antitumor and antimicrobial properties [44]. The other gene clusters found are likely to be involved with the biosynthesis of such products as phosphonoglycans, alkyl-O-dihydrogeranylmethoxyhydroquinones [45], and the antibiotics kanamycin [46], brasilicardin A and frankiamicin [47,48]. Interestingly, two bioclusters belonging to two classes I lanthipeptides and a class III lanthipeptide lacked any homology thereby providing further evidence that NP-BGCs are discontinuously distributed in the genomes of Micromonospora taxa [8,9]. Table 5. Identity of predicted natural product biosynthetic gene clusters using antiSMASH 6.0.0 alpha 1.

Genes Potentially Associated with Enviromental Stress
Stress-related genes detected in the genome of Dermacoccus abyssi strain MT1.1 T , an isolate from the same sediment sample as the M. provocatoris strain, gave clues to how this piezotolerant strain became adapted to environmental conditions which prevail in sea-floor sediment of the Challenger Deep of the Mariana Trench [49]. In the present study, the genome of M. provocatoris strain MT25 T annotated using NCBI Genbank [24][25][26] pipeline was seen to harbor genes associated with a range of stress responses, notably ones linked with carbon starvation, cold shock response, high pressure, osmoregulation and oxidative stress (Table S3), as was the case with the D. abyssi strain.
Deep-sea psychrophilic bacteria synthesize cold shock proteins essential for adaptation to low temperatures [50][51][52]. The genome of strain MT25 T contained genes predicted to encode cold shock proteins, as exemplified by genes clpB and hscB which are associated with the synthesis of ATP-dependent and Fe-S chaperones, respectively [52][53][54]. The genome also contains gene deaD encoding an RNA helicase involved in cold shock response and adaptation [55]. The strain has genes associated with the synthesis of branch-chain and long chain polysaturated fatty acids that are linked to membrane fluidity and functionality at low temperatures [49,52], including fabF, fabG, fabH and fabI genes which are responsible for the biosynthesis of β-ketoacyl-ACP synthase II, 3-oxoacyl-ACP reductase, ketoacyl-ACP synthase III, enoyl-ACP reductase and enoyl-ACP reductase, respectively (Table S3). The synthesis of low-melting point branched-chain and/or polyunsaturated fatty acids (PUFAs) is crucial as it allows organisms in cold environments to maintain membrane fluidity in a liquid crystalline state thereby allowing organisms to resist freeze-thaw cycles at low temperatures [56,57]. Low temperatures reduce enzymatic activity leading to the generation of reactive oxygen species (ROS). The genome of strain MT25 T contains genes sodN, trxA and trxB predicted to encode products that offset the harmful effects of superoxide dismutase, thioredoxin and thioredoxin-disulfide reductase respectively.
Bacteria living in deep-sea habitats have developed ways of dealing with osmotic stress, notably by synthesizing osmoregulators, these are small organic molecules (compatible solutes) induced under hyperosmotic stress [58][59][60]. In this context, strain MT25 T contains genes predicted to be involved in the biosynthesis of compatible solutes, such as opuA gene, which regulates the uptake of glycine/betaine thereby contributing to osmotic stress responses [61,62]. Similarly, genes asnO and ngg are predicted to be involved in the production of osmoprotectant NAGGN (n-acetylglutaminylglutamine amide) that has an important role in counteracting osmotic stress in deep-sea environments. It is produced by many bacteria grown at high osmolarity bacteria, such as Sinorhizobium meliloti [63].
Another consequence of high pressure on bacteria is that the transport of compounds, such as amino acids, is reduced leading to upregulation of transported molecules [64]. Genes associated with the production of different types of ABC transporter permeases were detected in strain MT25 T including branched-chain amino acid permeases that are upregulated at high pressure [65]. In addition, the genome of strain MT25 T contains pressure sensing and pressure adaptation genes, as illustrated by cycD, mdh and asd genes, which are linked to the production of a thiol reductant ABC exporter subunit, malate dehydrogenase and aspartate semialdehyde dehydrogenase, respectively. Similarly, secD and secF are predicted to encode protein translocase subunits and secG preprotein translocase unit [65,66], as shown in Table S3.
Bacteria able to grow in nutrient-limiting conditions need to store carbon compounds like glycogen [67]. In this respect, it is interesting that strain MT25 T contains a gene, gigA, which is predicted to encode glycogen synthase and another gene, gigx, which is linked with the production of a glycogen debranching enzyme responsible for the breakdown of this storage molecule. Furthermore, the strain has the capacity to produce carbonic anhydrase proteins which are required for fixation of carbon dioxide [65,68] thereby suggesting that its potential to grow as a lithoautotroph. This discovery provides further evidence that filamentous actinobacteria in carbon-limiting, extreme biomes are capable of adopting a lithoautotrophic lifestyle, as shown by the type strains of novel Blastococcus, Geodermatophilus and Modestobacter species [69][70][71][72][73].
Micromonosporae can grow under aerobic and microaerophilic conditions. Their ability to tolerate low oxygen tensions indicates an ability to grow in oxygen depleted biomes, such as lake and river sediments and soil prone to flooding [74,75]. Genome mining of strain MT25 T revealed many putative genes predicted to encode terminal oxidases involved in aerobic respiration, as witnessed by the cydB gene encoding cytochrome d ubiquinol oxidase subunit II, and genes ctad and coxb expressing cytochrome c oxidase subunits I and II, respectively. Several terminal dehydrogenase and reductase encoding genes involved in respiratory chains were detected, including ones predicted to express arsenate reductase arsc and ferredoxin reductase. Multiple genes predicted to encode succinate dehydrogenase used as electron donors under low oxygen conditions were also detected in the genome of strain MT25 T . Further support for the ability of the strain to adapt to different oxygen levels reflects its capacity to form cytochrome oxidase complexes that have different affinities for oxygen. Biological adaptations such as these may account for the presence of micromonosporae (including verrucosisporae) in marine habitats, including deep-sea sediments [2,3,76].

Microorganism
Micromonospora strain MT25 T was isolated from Mariana Trench sediment, sample no. 281, collected at a depth of 10,898 m (Challenger Deep; 11 • 19 911 N; 142 • 12 372 E) by the remotely operated submersible Kaiko, using a sterilized mud sampler, on 21 May 1998, during dive number 74. The sample was transported to the UK in an insulated container at 4 • C and stored at −20 • C until examined for actinobacteria. The test strain was isolated, purified and maintained using procedures described by Pathom-aree et al. [19]. M. tulbaghiae DSM 45142 T was maintained under the same conditions.

General Experimental Procedures
General Experimental Procedures. 1 H, 13 C, 15 N NMR experiments were recorded on a Bruker Avance 600 MHz NMR spectrometer AVANCE III HD (Billerica, MA, USA) equipped with a cryoprobe, in DMSO-d 6 . Low resolution electrospray mass spectra were obtained using a Perseptive Biosystems Mariner LC-MS (PerSeptive Biosystems, Framingham, MA, USA), and high-resolution mass data were generated on Finnigan MAT 900 XLT (Thermo-Finnigan, San Jose, CA, USA). HPLC separations were carried out using a Phenomenex reversed-phase (C 18 , 10 Å × 10 mm × 250 mm) column and an Agilent 1100 series gradient pump and monitored using an Agilent DAD G1315B variable-wavelength UV detector (Agilent Technologies, Waldbronn, Germany).

Fermentation Conditions
For the first-stage seed preparation, an agar grown culture of strain MT25 T , was inoculated into 10 mL of GYE medium (4.0 g glucose, 4.0 g yeast extract, agar 15 g, distilled H 2 O 1 L, pH 7.0). After 5 days incubation at 28 • C, with agitation, the first stage culture was used to inoculate the production fermentation, using ISP2 broth (yeast extract 4 g, malt extract 10 g, glucose 4 g, CaCO 3 2 g, distilled H 2 O 1 L, pH 7.3). The fermentation was incubated at 28 • C, with agitation, and the biomass was harvested on the seventh day. All media components were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Isolation and Purification of Secondary Metabolites
Harvested fermentation broth (6 L) was centrifuged at 3000 rpm for 20 min, and the HP20 resin together with the cell mass was washed with distilled water then extracted with MeOH (3 × 500 mL). The MeOH extracts were combined and concentrated under reduced pressure to yield 6.39 g solid extract. The extract was suspended in 250 mL of MeOH and then partitioned with n-hexane (3 × 250 mL). The remaining MeOH solubles were the sub-ject of further purification by Sephadex LH-20 column chromatography (CH 2 Cl 2 /MeOH 1:1) to yield 3 fractions. Final purification was achieved using reversed-phase HPLC (C 18 , 10 µm, 10 mm × 250 mm), employing gradient elution from 0-90% CH 3 CN/H 2 O containing 0.01% TFA over 40 min for fraction A (23 mg) to give compound 1 (16.2 mg) and fraction B (27 mg) and to give compound 2 (9.4 mg).

Phylogeny
An almost complete 16S rRNA gene sequence (1437 nucleotides) (Genbank accession number AY894337) was taken directly from the draft genome of the isolate using the ContEst16S tool from the EzBioCloud webserver (https://www.ezbiocloud.net/tools/ contest16s, accessed on 1 June 2018) [77]. The sequence was aligned with corresponding sequences of the most closely related type strains of Micromonospora species drawn from the EzBioCloud webserver [78] using MUSCLE software (Version No. 3.8.31, drive5, Berkeley, CA, USA) [79]. Pairwise sequence similarities were generated using the single gene tree option from the Genome-to-Genome Distance calculator (GGDC) webserver [37,80] and phylogenetic trees inferred using the maximum-likelihood [81], maximum-parsimony [82] and neighbor-joining [83] algorithms. A ML (maximum likelihood) tree was generated from alignments with RAxML (Randomized Axelerated Maximum Likelihood) [84] using rapid bootstrapping with the auto Maximum-Relative-Error (MRE) criterion [85] and a MP tree inferred from alignments with the tree analysis using the New Technology (TNT) program [86] with 1000 bootstraps together with tree-bisection-and-reconnection branch swapping and ten random sequence addition replicates. The sequences were checked for computational bias using the x 2 test taken from PAUP * (Phylogenetic analysis using parsimony) [87]. The trees were evaluated using bootstrap analyses based on 1000 replicates [88] from the MEGA X software package (Version No. 10.0.5, MEGA development team, State College, PA, USA) [89] and the two-parameter model of Jukes and Cantor, 1969 [90]. The 16S rRNA gene sequence of Catellatospora citrea IFO 14495 T (D85477) was used to root the tree.

Phenotypic Characterisation
The isolate was examined for a broad range of phenotype properties known to be of value in Micromonospora systematics [10,16]. Standard chromatographic procedures were used to detect isomers of diaminopimelic acid [91], whole-organism sugars [92] and polar lipids [93,94], using freeze dried biomass harvested from yeast extract-malt extract broth cultures (International Streptomyces Project [ISP] medium 2) [95]. Similarly, cellular fatty acids extracted from the isolate were methylated and analyzed using the Sherlock Microbial Identification (MIDI) system and the resultant peaks identified using the ACTINO 6 database [96].
Cultural and morphological properties of the isolate were recorded following growth on oatmeal agar (ISP medium 3) [95]. Growth from the oatmeal agar plate was examined for micromorphological traits using a scanning electron microscope (Tescan Vega 3, LMU instrument, Fuveau, France) and the protocol described by O'Donnell et al. [97]. The enzymatic profiles of strain MT25 T and M. tulbaghiae DSM 45142 T were determined using AP1-ZYM strips (bioMérieux) by following the instructions of the manufacture. Similarly, biochemical, degradation, physiological and staining properties were acquired using media and methods described by Williams et al. [98]. The ability of strain MT25 T to grow under different temperature and pH regimes and in the presence of various concentrations of sodium chloride were recorded on ISP2 agar as the basal medium; the pH values were determined using phosphate buffers. All of these tests were carried out using a standard inoculum of spores and mycelial fragments equivalent to 5.0 on the McFarland scale [99].
3.7. Whole-Genome Sequencing 3.7.1. DNA Extraction and Genome Sequencing Genomic DNA was extracted from wet biomass of a single colony of strain MT25 T following growth on yeast extract-malt extract agar for 7 days at 28 • C [95], using the modified CTAB method [100]. The sequence library was prepared using a NEB Next Fast DNA Fragmentation and Library Preparation Kit for an Ion Torrent (New England Biolabs, Hitchin, UK).
Briefly, the DNA sample (0.5 µg) was subjected to enzymatic fragmentation, end repaired and ligated to A1 and P2 adapters, followed by extraction of 490-500 bp fragments and PCR amplification. The PCR products were analyzed using a High Sensitivity DNA kit and BioAnalyser 2100 (Agilent Technologies LDA).
(UK Limited, Cheshire, UK). AMPure XP beads (Beckman Coulter, Brea, CA, USA) were used for DNA purification according to the protocol. The library was diluted to give a final concentration of 25 pM, and a template was prepared using an Ion PGM Hi-Q™ (Life Technologies Limited, Paisley, UK) View OT2 Kit and IonTorrent One Touch system OT2. The recovery of positive Ion Sphere Particles was achieved using the One Touch ES enrichment system. The sequencing reaction was conducted using an Ion PGM Hi-Q TM View Sequencing Kit, 316v2 chips and an IonTorrent PGM instrument with 850 sequencing flows, according to manufacturer's instructions (Life Technologies Limited, Paisley, UK), required for 400 nt read lengths.

Annotation of Genome and Bioinformatics
The sequencing reads were mapped onto reference genome sequences using CLC Genomics Workbench software (GWB, ver. 7.5, QIAGEN, LLC, Germantown, MD, USA). The reads were assembled using SPAdes v. 5.0.0.0 plugin (LifeTechnologies, Thermo Fisher Scientific, UK). The annotation of the genomic sequence was performed via NCBI GenBank annotation pipeline [24,101].

Detection of the Gene Clusters
The whole genome sequence of strain MT25 T was mined using AntiSMASH 6.0.0 alpha 1 ("Antibiotic and Secondary Metabolites Analysis Shell") [42] to detect biosynthetic gene clusters. The NCBI [24][25][26] GenBank annotation pipeline was used to detect the genes and proteins associated with bacterial adaptation.

GenBank Accession Number
This Whole Genome Shotgun sequence has been deposited at DDBJ/ENA/GenBank under accession number NZ_QNTW00000000. The version described in this paper is NZ_QNTW00000000.1.

Comparison of Genomes
The draft genome sequence of strain MT25 T was compared with corresponding sequences of the type strains of closely related Micromonospora strains, as shown in the phylogenomic analyses. A ML phylogenomic tree inferred using the codon tree option in the PATRIC webserver [102], based on aligned amino acids and nucleotides derived from 704 single copy core genes in the genome dataset matched against the PATRIC PGFams database (http://www.patricbrc.org, accessed on 10 July 2018), was generated using the RAxML algorithm [84]. Average nucleotide identity (ortho ANI) [103] and digital DNA-DNA hybridization [38] values were determined between the isolate and the type strains of M. aurantiaca, M. chalcea, M. marina and M. tulbaghiae, its closest phylogenomic neighbors.

Conclusions
Micromonospora strain MT25 T , an isolate recovered from sediment taken from the Mariana Trench in the Pacific Ocean, was shown to be most closely related to the type strain M. tulbaghiae following a genome-based classification. Characterization of strain MT25 T using a range of methods suggests that it belongs to a new Micromonospora species, which we name as Micromonospora provocatoris sp. nov. An associated bioassay-guided study together with structural analyses showed that the isolate has a potential to synthesize two major metabolites, n-acetylglutaminyl glutamine amide and desferrioxamine B. In line with previous studies on micromonosporae isolated from extreme habitats, strain MT25 T had a relatively large genome containing genes likely to be involved in the biosynthesis of novel natural products. Bioinformatic analyses of the genome of the M. provoactoris strain revealed a broad range of stress-related genes relevant to its survival in deep-sea sediments.