Pyomelanin-Producing Brevundimonas vitisensis sp. nov., Isolated From Grape (Vitis vinifera L.)

A novel endophytic bacterial strain, designated GR-TSA-9T, was isolated from surface-sterilized grape (Vitis vinifera L.). 16S rRNA gene sequence analyses showed that the isolate was grouped within the genus Brevundimonas, displaying the highest similarity with Brevundimonas lenta DS-18T (97.9%) and Brevundimonas kwangchunensis KSL-102T (97.8%) and less than 97.5% similarity with other members of Brevundimonas. The strain GR-TSA-9T was a gram negative, rod shaped, facultatively anaerobic, catalase and oxidase positive, and motile bacterium. Its growth occurred at 10–37°C (optimally 25–30°C), at pH 7.0–8.0, and in NaCl 0–1% (optimally 0%). It contained ubiquinone-10 as a respiratory quinone, and the major cellular fatty acids (>10% of the total) were C16:0 (14.2%) and summed feature 8 (C18:1ω7c and/or C18:1ω6c, 65.6%). The polar lipids present in the strain were phosphoglycolipids, phosphatidylglycerol, 1,2-di-O-acyl-3-O-[d-glucopyranosyl-(1→4)-α-d-glucopyranuronosyl]glycerol, and unidentified lipids (L1, L2, and L4). The strain had one 2,976,716bp circular chromosome with a G+C content of 66.4%. The digital DNA–DNA hybridization value between strain GR-TSA-9T and B. lenta DS-18T was 20.9%, while the average nucleotide identity value was 76.7%. In addition, the dDDH and ANI values to other members in this genus, whose genome sequences are available, are less than 21.1 and 77.6%. Genome annotation predicted the presence of some gene clusters related to tyrosine degradation and pyomelanin formation. Strain GR-TSA-9T produced a brown melanin-like pigment in the presence of L-tyrosine-containing media. The highest pigment production (0.19g/L) was observed in tryptic soy broth with 1.0mg/ml L-tyrosine at 25°C for 6days of culture. Biophysical characterization by ultraviolet (UV)–visible spectroscopy, Fourier-transform infrared spectroscopy, and electrospray ionization mass spectrometry confirmed that the pigment was pyomelanin. Additionally, melanized GR-TSA-9T cells could protect the cells against UVC exposure. The phylogenetic, genomic, phenotypic, and chemotaxonomic features indicated that strain GR-TSA-9T represents a novel melanin-producing species of Brevundimonas. The type strain was GR-TSA-9T (KCTC 82386T=CGMCC 1.18820T).


INTRODUCTION
The bacterial genus Brevundimonas was reclassified from the genus Pseudomonas as a member of the family Caulobacteraceae based on a polyphasic approach; type strains Brevundimonas diminuta ATCC 11568 T and Brevundimonas vesicularis ATCC 11426 T were proposed at the same time (Segers et al., 1994). Several Caulobacter species, such as C. bacteroides, C. intermedius, C. subvibrioides, C. variabilis, and Caulobacter subspecies, such as C. henricii subsp. aurantiacus, C. subvibrioides subsp. albus, as well as Mycoplana species, such as Mycoplana bullata (Abraham et al., 1999), were subsequently transferred to the genus Brevundimonas. As of April 2021, 32 species have been reported in this genus. 1 Members of the genus Brevundimonas survive in diverse environments, including deep-subsea floor sediment (Tsubouchi et al., 2013), different soil sources (Yoon et al., 2006(Yoon et al., , 2007Pham et al., 2016), the sea (Fritz et al., 2005), activated sludge (Ryu et al., 2007;Qu et al., 2019;Lee et al., 2020), humans (Sofer et al., 2007;Estrela and Abraham, 2010), aquatic habitats , and floor sediments (Tsubouchi et al., 2013(Tsubouchi et al., , 2014. The genus Brevundimonas has been reported to be a growth promoter in agriculture (Singh et al., 2016;Naqqash et al., 2020) and a bioremediation tool for the removal of copper (Rathi and Yogalakshmi, 2021), dimethachlon (Zhang et al., 2020), and arsenic toxicity (Singh et al., 2016). Furthermore, some species of this genus were found to be opportunistic pathogens isolated from clinical samples (Chandra et al., 2017;Ryan and Pembroke, 2018). Members of this genus are characterized as gramnegative, facultatively anaerobic or aerobic, rod-shaped, oxidase-and catalase-positive, non-fermenting bacteria with a relatively high DNA G + C content (Wang et al., 2012;Qu et al., 2019;Lee et al., 2020). Ubiquinone 10 (Q-10) is a major isoprenoid quinone. Interestingly, Brevundimonas sp. SGJ was reported to produce L-dihydroxylphenylalanine (DOPA) melanin, which is predominantly an indolic polymer that is widely used in cosmetics, agriculture, and medicine (Surwase et al., 2012a(Surwase et al., ,b, 2013. Melanin, a high-molecular-weight polymer, is a ubiquitous natural pigment widely encountered in all organisms. Most microbial melanins are formed through the transformation of either tyrosine (3, 4-DOPA pathway) or malonyl-coenzyme A (dihydroxynaphthalene pathway), which is facilitated by different sets of enzymes. The melanin precursor tyrosine is converted to L-DOPA by tyrosinase (EC 1.14.18.1) and laccase (EC 1.10.3.2;Pavan et al., 2020). The second pathway for melanin synthesis is, endogenously produced malonyl-CoA is catalyzed by polyketide synthases and converted to 1,3,6,8-tetrahydroxynaphthalene and polymerized to dihydroxynaphthalene melanin (Pavan et al., 2020). The third pathway for melanin synthesis is the homogentisate pathway, wherein 4-hydroxyphenylpyruvate dioxygenase (hppD; EC 1.13.11.27) is the most critical enzyme for transfer to homogentisic acid (HGA) melanins (or pyomelanin), and the hppD gene plays a vital role in the HGA pathway (Singh et al., 2018). 1 https://lpsn.dsmz.de/genus/brevundimonas In the present study, a new strain, GR-TSA-9 T , isolated from grapes was characterized as a novel species of the genus Brevundimonas based on the results obtained from a polyphasic taxonomic study. This strain is of interest because it can synthesize melanin. Whole-genome analysis was used to investigate the underlying pathways involved in pyomelanin biosynthesis mediated by the homogentisate pathway. Here, we present a novel species of the genus Brevundimonas, B. vitisensis sp. nov., with respect to taxonomy, genome analysis, and identification of pyomelanin production.

Bacterial Strains
Grapes were collected from Jeongeup (35°424.176' and 126°547.308'), and 5 g grape was surface-sterilized with 1.05% sodium hypochlorite for 10 min, followed by rinsing five times with sterile distilled water. After grinding in 20 ml sterile water, 100 μl of each sample was spread onto tryptic soy agar (TSA) medium and incubated at 25°C for 4 days. Single colonies were obtained and subsequently streaked on fresh TSA medium. One grayish white, circular, smooth, opaque, and flat isolate, designated as GR-TSA-9 T , was selected for further taxonomic study and preserved in sterile skimmed milk (10%, w/v) at −80°C. The strain was deposited at the Korean Collection for Type Cultures (KCTC) and the China General Microbiological Culture Collection (CGMCC) as KCTC 82386 T and CGMCC 1.18820 T , respectively. Unless otherwise stated, the cells were grown on TSA medium at 25°C for 5 days for subsequent tests.
Optical density at 600 nm (OD 600 ) was measured using an Optizen POP UV/VIS spectrophotometer (Optizen) to monitor the pH range and NaCl tolerance. The anaerobic growth was tested by incubating strain GR-TSA-9 T on TSA medium in an anaerobic chamber (Coy Scientific) filled with 86% N 2 , 7% CO 2 , and 7% H 2 at 25°C for 7 days. Other biochemical features, such as enzymes, were tested using API 20NE (bioMérieux) or API ZYM with NaCl 0.85% medium (bioMérieux) according to the manufacturer's protocol.

16S rRNA Gene Sequence Analysis
The almost-complete 16S rRNA gene sequence of strain GR-TSA-9 T was amplified by polymerase chain reaction using the universal primers 27F and 1492R (Lane, 1991), and sequencing was performed using primers 27F/1492R and 518F (5'-CCAGCAGCCGCGGTAATACG-3') and 800R (5'-TACCAGGGTATCTAATCC-3'). The nearly full-length 16S rRNA gene was 1394 nucleotides in length and was compared to the corresponding sequences of the type strains obtained from the EzBioCloud server . 2 BioEdit (V.7.0.5) was used to perform multiple sequence alignments using all validly published members of the family Caulobacteraceae. Phylogenetic trees were constructed using the neighbor-joining (NJ), minimum evolution (ME), and maximum likelihood (ML) algorithms with 1,000 bootstrap iterations in the Molecular Evolutionary Genetics Analysis version 7.0 program (Kumar et al., 2016). Evolutionary distances for the NJ, ME, and ML analyses were calculated using Kimura's two-parameter model.

Genomic Sequencing and Annotation
Genomic DNA of strain GR-TSA-9 T was isolated using a genomic DNA purification kit (MGmed, Republic of Korea). The quantity and quality of the extracted genomic DNA were measured using PicoGreen and Nanodrop (ratio A260/A280). Whole-genome sequencing was performed at the Macrogen facility (Macrogen, Korea) on the PacBio RSII (Pacific Biosciences, Inc.) and the Illumina sequencing platform and assembled using SMRT Portal (version 2.3) de novo assembler. Potential contamination of the genome sequence was verified using ContEst16S . 3 The assembled sequences were annotated using the National Center for Biotechnology Information Prokaryotic Genome Annotation Pipeline (PGAP) and the Rapid Annotation Subsystem Technology (RAST) server with the SEED database (Tatusova et al., 2016). Metabolic pathways were reconstructed using BlastKOALA based on the Kyoto Encyclopedia of Genes and Genomes pathway database (Kanehisa et al., 2016). The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strain GR-TSA-9 T and several closely related strains were calculated using the Genome-to-Genome Distance Calculation web server 4 using the BLAST method and recommended formula 2 (Meier- Kolthoff et al., 2013) as well as an ANI calculator. 5 The orthoANI values among the closely related strains were calculated using the standalone Orthologous Average Nucleotide Identity (OAT) software (Lee et al., 2016). Whole-genome sequences of closely related strains publicly available at NCBI GenBank were collected, and a whole-genome-based phylogenetic tree was constructed using the up-to-date bacterial core gene set and pipeline as described by Na et al. (2018). Briefly, the 92 core genes were extracted from genomes using Prodigal v2.6.3 (Hyatt et al., 2010) and hmmsearch v3.1b2 (Eddy, 2011). Predicted coding sequences (CDS) of 92 core genes were aligned by using Multiple Alignment using Fast Fourier Transform (MAFFT) for alignments (ver. 7.310; Katoh and Standley, 2013). Then, the phylogenomic tree was inferred by using the FastTree (Price et al., 2010) and viewed using MEGA v7.0 (Kumar et al., 2016). The branch support inference was based on 100 nonparametric bootstrap replicates, and the branch supports for the phylogenomic tree were evaluated using gene support index (GSI).

Chemotaxonomic Characterization
For fatty acid methyl ester analysis, cells grown for 3 days on TSA were extracted according to the instructions of the standard MIDI (Sherlock Microbial Identification System version 6.0), and cellular fatty acid content was analyzed by gas chromatography (Model 6890 N; Agilent) using the Microbial Identification software package (Sasser, 2006). Freeze-dried cells (100 mg) were used to extract isoprenoid quinones using a chloroform-to-methanol (2:1, v/v) mixture, followed by analysis using thin-layer chromatography as described by Collins et al. (1980). Subsequent analysis was performed using reverse-phase high-performance liquid chromatography with ultraviolet (UV) absorbance detection at 270 nm. Polar lipids were extracted from 100 mg of freeze-dried cells with a chloroform/methanol mixture (1:2, v/v) and then identified by two-dimensional thinlayer chromatography on Kieselgel 60 F254 plates (silica gel, 10cm × 10 cm; Merck). In addition, 0.2% ninhydrin (Sigma-Aldrich), α-naphthol, molybdenum blue (Sigma-Aldrich), 4% phosphomolybdic acid reagent, and Dragendorff 's solution were sprayed onto the plates to detect amino group-containing lipids, sugar-containing lipids, phosphorus-containing lipids, total lipids, and quaternary nitrogen-containing lipids, respectively.

Production and Purification of the Microbial Pigment
The melanin-producing ability of strain GR-TSA-9 T was confirmed by growth on TSA medium supplemented with 0 and 10 mg/ml of l-tyrosine followed by incubation at 25°C. Production was performed in TSB consisting of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg/ml l-tyrosine, and after inoculation with 1 ml of the cell suspension (OD 600 = 1.0) in 100 ml TSB, shaking at 25°C and 150 rpm for 1-7 days, and melanin production in the broth was measured at OD 400 using the standard synthetic melanin (Sigma-Aldrich, M8631) calibration curve method (Singh et al., 2018). Melanin was purified as previously described (El-Naggar and El-Ewasy, 2017). Briefly, the supernatant was centrifuged at 8,000 rpm to remove the cells, followed by adjustment of the pH of the supernatant to 2.0 using 6 M HCl; the samples were then allowed to stand for 4 h and centrifuged at 8,000 rpm to collect the precipitate. The melanin pellets were washed with distilled water three times and then centrifuged at 8,000 rpm for 10 min to obtain melanin. The purified melanin was freeze-dried for further use. In vitro-synthesized pyomelanin was produced by autooxidation of 10 mM HGA (Sigma-Aldrich, H0751) solution at pH 10 with constant stirring for 3 days as described by Schmaler-Ripcke et al. (2009).

UV-Visible Spectroscopy and Fourier-Transform Infrared Spectroscopy
To analyze the pigment content of GR-TSA-9 T , purified melanin was dissolved in 0.5 M NaOH solution, and the absorption value was recorded in the wavelength range of 200-1000 nm with a UV-visible spectrophotometer (Thermo Fisher Scientific, USA) by comparing it to a synthetic melanin standard. FT-IR spectroscopy was performed at the Center for Instrumental Analysis, Korea Basic Science Institute, Busan, Republic of Korea. The samples were pressed into disks under vacuum using a KBrpress. The FT-IR spectra of the KBr discs were recorded on an FT-IR Vertex 80v spectrophotometer (Bruker, USA). The spectra were read in the wavenumber region of 400-4000 cm −1 .

Electrospray Ionization Mass Spectrometry
Mass spectrometry analysis was performed using an API 32000 mass spectrometer (AB SCIEX, USA). Samples were dissolved in 1 M NaOH solution and then diluted with 50:50 methanol/ water. The optimized mass spectrometry parameters were as follows: curtain gas, 10; spray voltage, 4200; ion source gas, 20 psi; and flow rate, 10 μl/ml. Full spectra were collected in the m/z range of 50-1800 in the positive ion mode.

UVC Exposure and Sensitivity Tests
GR-TSA-9 T cells were cultured on TSA (non-melanized cells) and TSA with 10 mg/ml l-tyrosine (melanized cells) at 25°C for 48 h. The cells were washed twice with 1× phosphatebuffered saline. Next, 5 × 10 7 cells were irradiated with UVC (254 nm) at 450 mJ/cm, and 10 μl of aliquots from tenfold serial dilutions was spotted on TSA plates. All plates were incubated at 25°C for 3 days. The experiments were repeated at least twice, with similar results.

Phylogenetic Analyses
The almost-complete 16S rRNA gene amplicon of strain GR-TSA-9 T contained 1394 nucleotides and has been submitted to GenBank (accession number: MW442968.1). Based on 16S rRNA gene sequence analysis, strain GR-TSA-9 T appeared to belong to the genus Brevundimonas and was closely related to B. lenta DS-18 T (97.9%), B. kwangchunensis KSL-102 T (97.8%), and B. aurantiaca DSM 4731 T (97.5%), whereas it shared less than 97.5% similarity with other related species. Based on the novel species recognition threshold of 98.6% (Kim et al., 2014), strain GR-TSA-9 T was regarded as a novel species in the genus Brevundimonas. A phylogenetic tree was reconstructed using the NJ, ME, and ML methods. The results of these analyses suggest that strain GR-TSA-9 T belongs to the genus Brevundimonas. The genera Asticcacaulis, Caulobacter, and Phenylobacterium formed independent and monophyletic branches (Figure 1). Moreover, B. fluminis LA-55 T , B. viscosa CGMCC 1.10683 T , B. kwangchunensis KSL-102 T , B. lenta DS-18 T , and B. diminuta AJ 2068 T , the type species of genus Brevundimonas, were used to compare phenotypic properties and for chemotaxonomic analyses based on 16S rRNA similarity and phylogenetic tree analysis.

Genome Properties and Genetic Relatedness
The genome of strain GR-TSA-9 T was assembled using the SMRT Portal (version 2.3) de novo assembler, which is a single circular chromosome of 2,976,716 bp in size (the N50 value was 13,858 bp), with a coverage of 389X. By comparing two copies of the 16S rRNA gene fragment from the whole-genome sequence, a contaminating DNA sequence was not present in the genome assembly. The genomic GC content of GR-TSA-9 T was 66.4%, which was similar to that of other type strains in the genus Brevundimonas. According to the PGAP annotation, there were 2940 protein-coding genes and 56 RNA genes, including two 5S rRNA genes, two 16S rRNA genes, two 23S rRNA genes, four ncRNAs, and 46 tRNA genes. Cluster orthologous group (COG) annotation results showed that the functional categories of most coding sequences were classified as unknown (28.6% of total assigned COGs), general function prediction only (9.1% of the total assigned COGs), and amino acid transport and metabolism (5.8% of the total assigned COGs; Supplementary Figure 2).
The dDDH value between strain GR-TSA-9 T and the closest species B. lenta DS-18 T was 20.9%, while the ANI value was 76.7%. OrthoANI values based on the entire genome (Supplementary Figure 3) were 72.8-77.6% for the most closely related strains. Thus, strain GR-TSA-9 T was considered a distinct species of the genus Brevundimonas, considering the values obtained were significantly lower than the proposed dDDH (<70%) and ANI cutoff (95-96%) values for bacterial species delineation . The 92 core gene set-based Frontiers in Microbiology | www.frontiersin.org phylogenetic tree also supported strain GR-TSA-9 T forming a phylogenetic lineage within the genus Brevundimonas, consistent with the 16S rRNA-based phylogenetic tree (Figure 2).

Genome-Derived Features of Strain
GR-TSA-9 T Genome annotation was performed using the RAST server and BlastKOALA to reconstruct the metabolic pathway, and the following sections were predicted from genome sequences.

Motility
Genes related to the motility subsystem were detected in the whole genome, including basal-body rod protein FlgB, FlgC, and FlgD; flagellar hook-basal body complex protein FliE; flagellar motor switch protein FliM and FliN; flagellar L-ring protein FlgH; flagellar biosynthesis protein FlhB; FlhR and FlhA; and flagellar motor rotation proteins MotA and MotB. These data are consistent with the motility test results.

Respiration
Ferredoxin reductase, one of the anaerobic respiratory reductases, was annotated in the whole genome, which explains the fact that the strain can grow under anaerobic conditions. Among all respiration-related genes, most are related to electron-donating and electron-accepting reactions, followed by ATP synthases, biotin synthesis, the quinone oxidoreductase family, biogenesis of cytochrome C oxidases, biogenesis of c-type cytochromes, soluble cytochromes, and functionally related electron carriers.

Stress Response
Osmotic stress-related genes encoding enzymes involved in the synthesis of osmoregulated periplasmic glucans and osmoregulation were found in the whole genome, and no gene related to heat shock, cold shock, or salt stress was observed in the whole genome. These results explain why the strain grows at a limited temperature and salinity range.

Chemotaxonomic Characterization
The cellular fatty acid (>1%) composition of strain GR-TSA-9 T and five closely related strains is listed in Table 2; the most abundant fatty acids of strain GR-TSA-9 T were C 16:0 (14.2%) and summed featured 8 (65.6%). These results are consistent with those of closely related species within the genus Brevundimonas. However, the amount of some components, such as summed featured 3, C 17:1 w8c, and C 18:1 w7c 11-methyl, differs from the reference strains. The respiratory quinone detected in strain GR-TSA-9 T was Q-10, which was consistent with the genus Brevundimonas. The polar lipid profile of strain GR-TSA-9 T consisted of phosphoglycolipids, phosphatidylglycerol, 1,2-di-O-acyl-3-O-[d-glucopyranosyl-(1 → 4)-α-d-glucopyranuronosyl]glycerol, and unidentified Bootstrap values (>70%) calculated using the NJ, maximum likelihood (ML), and minimum evolution (ME) algorithms are shown. Filled circles on the nodes indicate that the relationships were also recovered by ML and ME algorithms, whereas open circles indicate nodes recovered by either ML or ME algorithms. Scale bar: 0.020 substitutions per nucleotide position.
lipids (L1, L2, and L4; Supplementary Figure 4), and lipid spots different from other closely related strains suggested that strain GR-TSA-9 T represents a novel species in the genus Brevundimonas.

Pigment Production
Strain GR-TSA-9 T was considered a potential melanin producer on TSA medium and can produce a higher amount of melanin in l-tyrosine-containing medium than without l-tyrosine (Figures 3A,B). On TSA with l-tyrosine, the synthesis of brown pigment was initiated at 3 d and the pigment increased to brown, dark brown, and blackbrown at 4, 5, and 6 days, respectively. Melanin production in TSB was observed after 3 days of shaking, and at 6 days, the highest yield of melanin (0.19 g/L) was obtained ( Figure 3C).
A B C FIGURE 3 | Melanin-like pigment production of strain GR-TSA-9 T on tryptic soy agar (TSA) medium. (A) Melanin-like pigment production of strain GR-TSA-9 T on TSA without and with L-tyrosine participation; dark brown pigment was produced on the L-tyrosine medium for 5 days. (B,C) Effect of L-tyrosine concentration on melanin production in TSB medium. The graph represents the average of the results from three replicate measurements.
Frontiers in Microbiology | www.frontiersin.org -Barrau et al., 2004;Rodríguez-Rojas et al., 2009;Orlandi et al., 2015;Hocquet et al., 2016;Zeng et al., 2017;Kurian and Bhat, 2018;Bolognese et al., 2019). This suggests that encoding the most critical enzymes 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27), araT (EC 2.6.1.57), and other enzymes such as hmgR annotated in the whole genome demonstrated that the HGA pathway is the melanogenic pathway in strain GR-TSA-9 T ( Figure 4B). In addition, we found brown or black pigments in some of the most closely related strains such as B. Based on these analyses, we suggest that the brown-to-black pigment production in many species in the genus Brevundimonas is because of the production of pyomelanin via the HGA pathway rather than the L-DOPA pathway.

Pigment Characterization
The wavelength of maximum absorbance was scanned in the range of 200-1000 nm. The wavelength of maximum absorbance of the extracted pigment from the culture supernatant and synthetic melanin standard was observed at 210 nm (Supplementary Figure 5), which is a typical feature of melanin (ranges between 196 and 300 nm; Surwase et al., 2013;Zeng et al., 2017). The FT-IR spectra were analyzed to confirm that the extracted bacterial pigment was pyomelanin type of melanin. The FT-IR spectra of the extracted bacterial pigment and synthetic pyomelanin standard are shown in Figure 5A. The spectra were recorded at 4000-400 cm −1 using an FT-IR spectrophotometer. Both spectra revealed a broad absorption peak at 3650-3100 cm −1 , corresponding to the ▬OH group and ▬NH group. The peaks at 3000-2900 and 1250-1200 cm −1 were attributed to C▬H groups. The strong absorption peak observed at 1450-1770 cm −1 was ascribed to the C〓C and C〓O groups. The peak between 1250 and 1200 cm −1 corresponds to the N▬H group and C▬N (secondary amine). The high degree of resemblance in the main absorption peaks indicated that the pigment isolated from strain GR-TSA-9 T was pyomelanin. Synthetic pyomelanin and purified bacterial melanin were analyzed by ESI-MS. In addition, nine prominent peaks were observed in the mass spectrum of synthetic pyomelanin with m/z values of 96.5, 121.4, 135.5, 141.4, 167.5, 181.5, 223.6, 239.6, and 263.4 ( Figure 5B). These nine peaks were also detected in the mass spectrum of purified bacterial melanin ( Figure 5C). In particular, m/z 141.4 and 263.4 peaks were also detected in a previous report (Singh et al., 2018). These results suggest that the purified bacterial melanin is similar to synthetic pyomelanin.

UVC Tolerance
To test the photoprotective property of bacterial melanin, cells were irradiated with short-wavelength UVC, which is a damaging type of UV radiation. UVC is absorbed by DNA, resulting in the formation of pyrimidine adducts and strand breaks (Rastogi et al., 2010). The cultured cells on TSA (non-melanized cells) and TSA with 10 mg/ml l-tyrosine (melanized cells) were irradiated with UVC. The melanized cells grew better than the non-melanized cells after UVC irradiation (Figure 6). This finding suggests that cells grown in melanin-containing media showed significantly higher resistance to UV radiation than those grown on TSA medium alone.
FIGURE 6 | Photoprotective properties of strain GR-TSA-9 T . Non-melanized and melanized GR-TSA-9 T cells were irradiated with 450 mJ/cm 2 UVC and then spotted in serial dilutions on TSA media for 3 days.

CONCLUSION
Together, the results, including phenotypic, physiological, phylogenetic, and biochemical analyses, indicate that strain GR-TSA-9 T represents a novel member of the genus Brevundimonas. The phylogenetic relationships of strain GR-TSA-9 T and other type strains of the genus Brevundimonas were revealed by phylogenetic trees based on 16S rRNA and core gene sets from the whole-genome sequence. The similarities in physiological characteristics and differences in biochemical characteristics with the closely related strains suggest that GR-TSA-9 T is a new member of the genus Brevundimonas. The present study is the first to report a type strain in the genus Brevundimonas, which produces pyomelanin. This study provides new insights into the formation and regulation mechanism of melanin, provides the exploration potential of strain GR-TSA-9 T , and paves the way for industrial production in the future. The isolated melanin may have potential applications in the cosmetic and pharmaceutical industries.
The accession numbers for 16S rRNA and the whole genome of strain GR-TSA-9 T are MW442968.1 and CP067977.

DATA AVAILABILITY STATEMENT
The datasets generated for this study are available in the NCBI database. The accession numbers for 16S rRNA and the whole genome of strain GR-TSA-9 T are as follows: GenBank MW442968.1 and CP067977.