Actinoplanes aureus sp. nov., a novel protease-producing actinobacterium isolated from soil

A novel protease-producing actinobacterium, designated strain NEAU-A11T, was isolated from soil collected from Aohan banner, Chifeng, Inner Mongolia Autonomous Region, China, and characterised using a polyphasic approach. The hydrolytic enzymes, such as proteases, played critical roles in destruction of fungi by degrading the protein linkages to disrupt integrity in the cell wall. This suggested that the isolate could be a good biocontrol candidate against pathogens to control fungal diseases. On the basis of 16S rRNA gene sequence analysis, strain NEAU-A11T was indicated to belong to the genus Actinoplanes and was most closely related to Actinoplanes rectilineatus JCM 3194 T (98.9%). Cell walls contained meso-diaminopimelic acid as the diagnostic diamino acid and the whole-cell sugars were arabinose, xylose and glucose. The phospholipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and two phosphatidylinositol mannosides. The predominant menaquinones were MK-9(H4), MK-9(H6) and MK-9(H8). The major fatty acids were C18:0, C16:0, C18:1ω9c, C17:0 and C15:0. Genome sequencing revealed a genome size of 10,742,096 bp, a G + C content of 70.5% and 9,514 protein-coding genes (CDS), including 102 genes coding for protease. Moreover, Genome analysis showed that strain NEAU-A11T contained 255 glycoside hydrolases (GHs), 152 glycosyl transferases (GTs), 40 carbohydrate esterases (CEs), 26 polysaccharide lyases (PLs), and 12 auxiliary activities (AAs) genes. Genome mining analysis using antiSMASH 5.0 led to the identification of 20 putative gene clusters responsible for the production of diverse secondary metabolites. Phylogenetic analysis using the 16S rRNA gene sequences showed that the strain formed a stable clade with A. rectilineatus JCM 3194 T in the genus Actinoplanes. Whole-genome phylogeny showed strain NEAU-A11T formed a stable phyletic line with Actinoplanes lutulentus DSM 45883 T (97.6%). However, whole-genome average nucleotide identity value between strain NEAU-A11T and its reference strains A. rectilineatus JCM 3194 T and A. lutulentus DSM 45883 T were found to be 81.1% and 81.6%, respectively. The levels of digital DNA-DNA hybridization between them were 24.6% (22.2–27.0%) and 24.8% (22.5–27.3%), respectively. The values were well below the criteria for species delineation of 70% for dDDH and 95–96% for ANI, suggesting that the isolate differed genetically from its closely related type strain. The content of G + C in genomic DNA was 70.5%, within the range of 67–76%. In addition, evidences from phenotypic, chemotaxonomic and genotypic studies indicated that strain NEAU-A11T represents a novel species of the genus Actinoplanes, for which the name Actinoplanes aureus sp. nov. is proposed, with NEAU-A11T (= CCTCC AA 2019063 T = JCM 33971 T) as the type strain.

A. lutulentus DSM 45883 T were found to be 81.1% and 81.6%, respectively. The levels of digital DNA-DNA hybridization between them were 24.6% (22.2-27.0%) and 24. 8% (22.5-27.3%), respectively. The values were well below the criteria for species delineation of 70% for dDDH and 95-96% for ANI, suggesting that the isolate differed genetically from its closely related type strain. The content of G ? C in genomic DNA was 70.5%, within the range of 67-76%. In addition, evidences from phenotypic, chemotaxonomic and genotypic studies indicated that strain NEAU-A11 T represents a novel species of the genus Actinoplanes, for which the name Actinoplanes aureus sp. nov. is proposed, with NEAU-A11 T (= CCTCC AA 2019063 T = JCM 33971 T ) as the type strain.

Introduction
The genus Actinoplanes was first proposed by Couch (1950) as a member of the family Micromonosporaceae, with Actinoplanes philippinensis as the type species. Currently, there are 47 species with validly published and correct names (https://lpsn. dsmz.de/genus/actinoplanes), including the latest described species Actinoplanes deserti (Habib et al. 2018). Actinoplanes species are abundant sources of bioactive compounds (Parenti and Coronelli 1979;Simone et al. 2013;Iorio et al. 2014;Rückert et al. 2014;Huang et al. 2015), including antibiotics with therapeutic value such as teicoplanin (Sosio et al. 2004), and the alpha-glucosidase inhibitor acarbose which is a potent drug used worldwide in the treatment of type 2 diabetes (Schwientek et al. 2012 (Hayakawa et al. 1991). This process is known as dehiscence (Uchida et al. 2011;Higgins 1967). Goodfellow et al. (1990) provided the detailed phenotypic and chemotaxonomic analysis of the genus, and a comprehensive phylogenetic analysis of the genus was given by Tamura and Hatano (2001). The peptidoglycan of members within this genus contains meso-diaminopimelic acid, while, hydroxyldiaminopimelic acid can also be present. The diagnostic sugar of whole-cell hydrolysates is xylose, moreover, small amounts of galactose and/or arabinose are also found. Phosphatidylethanolamine is the diagnostic phospholipid. The G ? C contents of the genomic DNA vary from 67 to 76% (Sazak et al. 2012).
In this study, we described a novel species, protease-producing strain NEAU-A11 T from continuous cropping soil of cucumbers collected from Aohan Banner, Chifeng, Inner Mongolia Autonomous Region, China. It produced protease involving in destroying the components of fungal cell wall, which was an important mechanism of the biocontrol of phytopathogenic fungi (Deng et al. 2018). The isolate had the potential to be a eco-friendly antagonistic microorganism used as biocontrol agents against plant pathogens without adverse effects as chemical fungicides. In this study, we reported on polyphasic taxonomy of strain NEAU-A11 T and proposed this strain represents a new species of the genus Actinoplanes.

Materials and methods
Isolation and maintenance of the organism Strain NEAU-A11 T was isolated from soil collected from Aohan banner, Chifeng, Inner Mongolia Autonomous Region, China (42°29 0 N, 119°92 0 E). Before isolation, the soil sample was air-dried at room temperature for 14 days. For isolation, 5 g of dried soil was diluted in sterile distilled water (45 ml), and then the soil suspension was incubated in a constant temperature shaker incubator at 28°C and 250 g for 30 min. Subsequently, the strain was isolated using the standard dilution plate method and grown on sodium succinate-asparagine agar (Piao et al. 2017) supplemented with cycloheximide (50 mg l -1 ) and nalidixic acid (20 mg l -1 ). After 3 weeks of aerobic incubation at 28°C, colonies were transferred and purified on oatmeal agar [International Streptomyces Project (ISP) medium 3] (Shirling and Gottlieb 1966) and maintained as glycerol suspensions (30%, v/v) at -80°C.

Phenotypic characterisation
The cultural characteristics of strain NEAU-A11 T were determined after growing for 14 days at 28°C on various ISP (ISP 1-7) media (Shirling and Gottlieb 1966), Bennett's agar (Waksman 1967), Czapek's agar (Jones 1949) and nutrient agar (Jones 1949). ISCC-NBS color charts (Kelly 1964) were used to determine colours of aerial and substrate mycelia. Morphological characteristics were observed by light microscopy (Nikon ECLIPSE E200) and scanning electron microscopy (Hitachi SU8010) using cultures grown on ISP 3 agar at 28°C for 4 weeks as previously described method (Jin et al. 2019). The Gram reaction was performed according to the protocol of Gregersen (1978) by using KOH for cell lysis. Spore motility was identified by light microscopy (Nikon ECLIPSE E200) observation of cells suspended in 1 mM phosphate buffer (pH 7.0). Growth at different temperatures (4, 10, 15, 18, 25, 28, 32, 35, 37, 40 and 42°C) was determined on ISP 3 agar after incubation for 14 days. The pH range for growth (pH 3.0-12.0, at intervals of 1 pH units) was tested in Glucose-yeast extract broth (GY) (Jia et al. 2013) using the buffer system: pH 4.0-5.0, 0.1 M citric acid/ 0.1 M sodium citrate; pH 6.0-8.0, 0.1 M KH 2 PO 4 / 0.1 M NaOH; pH 9.0-10.0, 0.1 M NaHCO 3 /0.1 M Na 2 CO 3 ; pH 11.0-12.0, 0.2 M KH 2 PO 4 /0.1 M NaOH (Cao et al. 2020;Zhao et al. 2019). NaCl tolerance (0-12%, with an interval of 1%, w/v) for growth were tested after 14 days growth in GY broth at 28°C. The utilization of sole carbon and nitrogen sources, decomposition of cellulose, hydrolysis of starch and aesculin, reduction of nitrate, coagulation and peptization of milk, liquefaction of gelatin and production of H 2 S were examined as described previously (Gordon et al. 1974;Yokota et al. 1993). Hydrolysis of Tweens (20, 40 and 80) and production of urease were tested as described by Smibert and Krieg (1994). The related type strain was also included for comparison in all tests. In addition, the protease was detected by observing whether the agar plate (5% skimmed milk powder, 2% agar, sterilized at 121°C) produced clear rings.

Chemotaxonomic analyses
For the chemotaxonomic analysis, freeze-dried biomass was prepared from cultures grown in GY medium on a rotary shaker (250 g) at 28°C for 7 days. The isomer of diaminopimelic acid (DAP) in the cell wall hydrolysates was derivatized and analysed by a HPLC method (McKerrow et al. 2000). The whole-cell sugars were performed according to the procedures developed by Lechevalier and Lechevalier (1980). The phospholipids in cell were separated by two-dimensional TLC and identified using the method of Minnikin et al. (1984). Menaquinones were extracted from freeze-dried biomass and purified according to Collins (1985). Extracts were analysed by a HPLC-UV method (Wu et al.1989) using an Agilent Extend-C 18 Column (150 9 4.6 mm, i.d. 5 lm), typically at 270 nm. The mobile phase was acetonitrile/propyl alcohol (60:40, v/v) . The presence of mycolic acids was checked by the acid methanolysis method of Minnikin et al. (1980). To determine cellular fatty acid compositions, strain NEAU-A11 T and its closely related strain were cultivated in GY medium in shake flasks at 28°C for 7 days. Fatty acid methyl esters were extracted from the biomass as described by Gao et al. (2014a) and analysed by GC-MS using the method of Xiang et al. (2011).
DNA preparation, amplification and determination of 16S rRNA sequences Extraction of chromosomal DNA and PCR amplification of the 16S rRNA gene sequence were carried out according to the procedure developed by Kim et al. (2000). The PCR product was purified and cloned into the vector pMD19-T (Takara) and sequenced using an Applied Biosystems DNA sequencer (model 3730XL). The almost full-length 16S rRNA gene sequence of strain NEAU-A11 T , comprising 1480 bp, was obtained and compared with type strains available in the EzBioCloud server (https://www.ezbiocloud. net/) (Yoon et al. 2017a) and retrieved using NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi;), and then submitted to the GenBank database. The phylogenetic trees were constructed based on the 16S rRNA gene sequences of strain NEAU-A11 T and related reference species. Multiple sequences were aligned in Molecular Evolutionary Genetics Analysis (MEGA) software version 7.0 using the Clustal W algorithm and trimmed manually where was necessary. Phylogenetic trees were constructed with maximum likelihood (Felsenstein 1981) and neighbourjoining (Saitou and Nei 1987) algorithms using MEGA 7.0 (Kumar et al. 2016). The stability of the topology of the phylogenetic tree was assessed using the bootstrap method with 1000 repetitions (Felsenstein 1985). A distance matrix was generated using Kimura's two-parameter model (Kimura 1980). All positions containing gaps and missing data were eliminated from the dataset (complete deletion option). 16S rRNA gene sequence similarities between strains were calculated on the basis of pairwise alignment using the EzBioCloud (Yoon et al. 2017a).
Genomic analysis, DNA-DNA hybridization and DNA G ? C content The genomic DNA of strain NEAU-A11 T was extracted by SDS method for genome sequencing and assembly. The harvested DNA was detected by the agarose gel electrophoresis and quantified by Qubit.
Whole-genome sequencing was performed on the Illumina HiSeq PE150 platform. A-tailed, ligated to paired-end adaptors and PCR amplified with a 350 bp insert was used for the library construction at the Beijing Novogene Bioinformatics Technology Co., Ltd. Illumina PCR adapter reads and low quality reads from the paired-end were filtered by the step of quality control using our own compiling pipeline. All good quality paired reads were assembled using the SOAP denovo (Li et al. 2010(Li et al. , 2008 (http://soap.genomics. org.cn/soapdenovo.html) into a number of scaffolds. Then the filter reads were handled by the next step of the gap-closing. Whole-genome phylogeny was generated using TYGS server (http://tygs.dsmz.de) which is free, publicly available on the Internet as an easy-tocomprehend interface for submitting requests and browsing results. An according menu item allows for the rapid submission of a request including an e-mail address and one to several user-defined genomes. (Meier-Kolthoff and Göker 2019). The dDDH and ANI values were determined between the genomes of strain NEAU-A11 T and A. rectilineatus JCM 3194 T and A. lutulentus DSM 45883 T online at http://ggdc. dsmz.de using the Genome-to-Genome Distance Calculation (GGDC 2.1) (Meier-Kolthoff et al. 2013) and the ChunLab's online ANI Calculator (www. ezbiocloud.net/tools/ani) (Yoon et al. 2017b), respectively. The DNA G ? C content was calculated from the genome sequence. ''Antibiotics and secondary metabolite analysis shell'' (antiSMASH) version 5.0 was employed to analyze the bioactive secondary metabolites (Blin et al. 2019).

Phenotypic characteristics
Morphological observation of 4-week-old culture of strain NEAU-A11 T grown on ISP 3 medium showed that it had the typical characteristics of the genus Actinoplanes. Strain NEAU-A11 T was observed to produce branched, non-fragmenting substrate hyphae. The sporangia were 4.6-8.7 lm in size (Fig. S1) with irregular shape similar to Actinoplanes nipponensis (Wink et al. 2014), Actinoplanes lichenis (Phongsopitanun et al. 2016) and Actinoplanes luteus (Suriyachadkun et al. 2015). The sporangiospores were motile. Strain NEAU-A11 T was found to grow well on ISP 2, ISP 3, ISP 4, ISP 7 and nutrient agar media; grow moderately on ISP 6 medium; grow poorly on ISP 1, ISP 5 and Czapek's agar media; but no growth occurred on Bennett's agar medium. Moderate olive and strong yellow pigments were observed on ISP 6 and nutrient agar, respectively. The summary of cultural characteristics of strain NEAU-A11 T and its phylogenetic neighbors were shown in Table S1 for comparative analysis. The strain was found to grow at a temperature range of 18-37°C (optimum temperature 28°C), pH 6.0-9.0 (optimum pH of 7.0) and NaCl tolerance of 0-4%. The physiological and biochemical properties of strain NEAU-A11 T are shown in Table 1.

Molecular characteristics
The almost complete 16S rRNA gene sequence of strain NEAU-A11 T (1480 bp) was determined and deposited with the accession number MW272536 in the GenBank/EMBL/DDBJ databases. Comparative 16S rRNA gene sequence analysis using the EzBio-Cloud server showed that strain NEAU-A11 T belongs to the genus Actinoplanes and is closely related to A. rectilineatus JCM 3194 T (98.9%). Phylogenetic analysis using the 16S rRNA gene sequence showed that the strain formed a stable clade with A. rectilineatus JCM 3194 T in the neighbour-joining tree (Fig. 1). This relationship was also observed in the maximum-likelihood tree (Fig. S3). However, wholegenome phylogeny showed strain NEAU-A11 T formed a stable phyletic line with A. lutulentus DSM 45883 T (97.6%) (Fig. 2). Based on the 16S rRNA gene sequence similarities and phylogenic analysis, A. rectilineatus JCM 3194 T and A. lutulentus DSM 45883 T were selected as the reference strains for comparative analysis.
The assembled genome sequence of strain NEAU-A11 T was found to be 10,742,096 bp long, composed of 109 scaffolds with an N50 of 187,467 bp and a DNA G ? C content of 70.5%. The draft genome sequence was deposited at the GenBank/EMBL/DDBJ under the accession number JADQTO000000000. NCBI Prokaryotic Genome Annotation Pipeline (PGAP) revealed four copies of the 5S rRNA genes, Table 1 Differential characteristics of strain NEAU-A11 T and its closely related species of the genus Actinoplanes  Table S2. Genome analysis showed that strain NEAU-A11 T contained 255 glycoside hydrolases (GHs), 152 glycosyl transferases (GTs), 40 carbohydrate esterases (CEs), 26 polysaccharide lyases (PLs), and 12 auxiliary activities (AAs) genes. Meanwhile, it was also showed that strain NEAU-A11 T contained 303 biosynthesis genes of antibiotics, 18 prodigiosin biosynthesis genes, 17 streptomycin biosynthesis genes, 8 biosynthesis genes of enediyne antibiotics, 6 penicillin and cephalosporin biosynthesis genes, 5 biosynthesis genes of ansamycins, 4 biosynthesis genes of vancomycin group antibiotics, 4 novobiocin biosynthesis genes, 3 acarbose and validamycin biosynthesis genes, 2 carbapenem biosynthesis genes, 2 isoquinoline alkaloid biosynthesis genes, 2 neomycin, kanamycin and gentamicin biosynthesis genes and 1 flavonoid biosynthesis gene. Genome mining analysis using antiSMASH 5.0 led to the identification of b Fig. 1 Neighbour-joining tree based on 16S rRNA gene sequences showing relationships between strain NEAU-A11 T (1480 bp) and the 47 phylogenetically closely related representative species with validly-published names in the genus Actinoplanes. The out-group used was Dactylosporangium aurantiacum DSM 43157 T . Only bootstrap values above 50% (percentages of 1000 replications) are indicated. Asterisks indicate branches also recovered in the maximum-likelihood tree; Bar, 0.005 nucleotide substitutions per site Fig. 2 Whole-genome sequence tree generated with TYGS for strain NEAU-A11 T and closely related species of the genus Actinoplanes. Th e out-group used was Dactylosporangium aurantiacum DSM 43157 T . Tree inferred with FastME from GBDP distances calculated from genome sequences. Branch lengths are scaled in terms of GBDP distance formula d5; numbers above branches are GBDP pseudo-bootstrap support values from 100 replications 20 putative gene clusters responsible for the production of diverse secondary metabolites. There were 17 gene clusters displayed very low similarities to the known gene clusters of xantholipin, azinomycin B, dynemicin A, oligomycin, diazepinomicin, himastatin, azalomycin F3a, mediomycin A and so on. Therefore, strain NEAU-A11 T has great potential to produce novel bioactive compounds. The other three biosynthetic gene clusters with more than 50% similarities to the known clusters were related to the production of alkyl-O-dihydrogeranyl-methoxyhydroquinones, citrulassin E and desferrioxamin B. Especially, one putative gene cluster shared 100% similarity to the reported type III polyketide biosynthetic gene cluster of alkyl-O-dihydrogeranylmethoxyhydroquinones in Actinoplanes missouriensis (Awakawa et al. 2011). Hence, it is speculated that strain NEAU-A11 T had significant potential to be a rich source for producing various bioactive compounds. In addition, 102 protease (such as YP_003344293, YP_008738346, YP_003836524, YP_007213461, YP_003300042) genes were identified in the genome of NEAU-A11 T . Moreover, the results of agar plates bioassay showed that strain NEAU-A11 T had the ability to produce protease (Fig. S4). Proteases may be involved in the biological control of plant-pathogenic fungi because they may play an important role in fungal cell wall lysis . Therefore, strain NEAU-A11 T is a potential biological control agent. Digital DNA-DNA hybridization and ANI values were employed to further clarify the relatedness between strain NEAU-A11 T and A. rectilineatus JCM 3194 T and A. lutulentus DSM 45883 T . The levels of digital DNA-DNA hybridization between them were 24.6% (22.2-27.0%) and 24.8% (22.5-27.3%), respectively, which was below the threshold value of 70% recommended by Wayne et al. (Wayne et al. 1987) for assigning strains to the same genomic species. Similarly, the low ANI values between strain NEAU-A11 T and its reference strains A. rectilineatus JCM 3194 T and A. lutulentus DSM 45883 T were found to be 81.1% and 81.6%, respectively, a result below the threshold used to delineate prokaryote species (Richter and Rossello-Mora 2009;Chun and Rainey 2014).
Beside of the genotypic evidence above, strain NEAU-A11 T also can be distinguished from its closely related strains by phenotypic characteristics (Table 1). Differential cultural characteristics contain: NaCl tolerance of strain NEAU-A11 T is up to 4% (w/ v), which is higher than that of A. rectilineatus JCM 3194 T (3%); and strain NEAU-A11 T could grow at the maximum temperature of 37°C, which is lower than that of A. rectilineatus JCM 3194 T (42°C) and higher than that of A. lutulentus DSM 45883 T (32°C). Other phenotypic differences include: coagulation and peptization of milk, hydrolysis of Tweens (40 and 80) and aesculin, production of H 2 S and utilization of D-mannitol, D-mannose, D-galactose, Dsorbitol, inositol, lactose, L-alanine, L-glutamine, Lproline, L-serine, L-threonine and maltose. The content of G ? C in genomic DNA is 70.5%, within the range of 67-76%. Both ANI and dDDH values are below the proposed and generally accepted species boundaries. In addition, protease secreted by strain NEAU-A11 T may contributes to antagonism against fungal pathogens via degradation and destruction of the cell wall, indicating the isolate could be used as an biological control agent against pathogenic fungus. Therefore, based on a combination of chemotaxonomic, morphological, molecular and physiological data, strain NEAU-A11 T represents a novel species of the genus Actinoplanes, for which the name Actinoplanes aureus sp. nov. is proposed.
Description of Actinoplanes aureus sp. nov.
Actinoplanes aureus (au're.us. L. masc. adj. aureus golden) An aerobic, Gram-stain positive, protease-producing actinobacterium that forms well-developed substrate mycelia carrying irregular sporangia (4.6-8.7 lm). The sporangiospores are motile. Grows well on ISP 2, ISP 3, ISP 4, ISP 7 and nutrient agar media; grows moderately on ISP 6 medium; grows poorly on ISP 1, ISP 5 and Czapek's agar media; but no growth occurs on Bennett's agar medium. Moderate olive and strong yellow pigments are observed on ISP 6 and nutrient agar, respectively. Growth occurs at pH 6.0-9.0 (optimum pH 7.0), temperature range of 18-37°C (optimum 28°C) and in the maximum presence of 4% NaCl (w/v). Positive for hydrolysis of aesculin and starch and production of H 2 S, but negative for hydrolysis of Tweens (20, 40 and 80), liquefaction of gelatin, production of urease, coagulation and peptization of milk, decomposition of cellulose and reduction of nitrate. Detailed utilization of carbon and nitrogen sources are referred to Table S3. Cell walls contain meso-diaminopimelic acid as the diagnostic diamino acid and the whole-cell sugars are arabinose, xylose and glucose. The phospholipid profile contains diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and two phosphatidylinositol mannosides. The menaquinones are MK-9(H 8 ), MK-9(H 6 ) and MK-9(H 4 ). The predominant cellular fatty acids ([ 10%) are C 18:0 , C 16:0 , C 18:1 x9c, C 17:0 and C 15:0 . The DNA G ? C content of the type strain is 70.5%.
The type strain is NEAU-A11 T (= CCTCC AA 2,019,063 T = JCM 33971 T ), isolated from soil collected from Aohan banner, Chifeng, Inner Mongolia Autonomous Region, China. The GenBank/EMBL/ DDBJ accession number for the 16S rRNA gene sequence of strain NEAU-A11 T is MW272536. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JADQTO000000000. The version described in this paper is version JADQTO000000000.1.
Acknowledgements We thank Prof. Aharon Oren for his valuable help with naming the species.
Author contributions JS performed the laboratory experiments, designed the experiments and revised the manuscript. XS performed the laboratory experiments, analysed the data, and drafted the manuscript. XL contributed to the biochemical characterisation. CH and ZH contributed to the polyphasic taxonomy. JZ contributed to the fatty acids determination. BH and XD contributed to the morphological analyses. XW and WX participated to the discussions of each section of experiments, designed the experiments and revised the manuscript. Data availability The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain NEAU-A11 T is MW272536. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JADQTO000000000. The version described in this paper is version JADQTO000000000.1.

Declarations
Conflict of interest The authors declare that they have no conflict of interest.
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