Massilia Cellulosiltytica Sp. Nov., A Novel Cellulose-degrading Bacterium Isolated from Rhizosphere Soil of Rice (Oryza Sativa L.) and whole Genome Analysis

A bacterial strain, Gram-stain negative, rod-shaped, aerobic and cellulose-degrading, designated NEAU-DD11 T , was isolated from rhizosphere soil of rice collected from Northeast University in North-east China. Base on 16S rRNA gene sequence analysis, strain NEAU-DD11 T belongs to the genus Massilia and shared high sequence similarities with Massilia phosphatilytica 12-OD1 T (98.46 %) and Massilia putida 6NM-7 T (98.41 %). Phylogenetic analysis based on the 16S rRNA gene and whole genome sequences indicated that strain NEAU-DD11 T formed a stable cluster with M. phosphatilytica 12-OD1 T and M. putida 6NM-7 T . The major fatty acids of the strain were C 16:0 , C 17:0 -cyclo and C 16:1 ω7c. The respiratory quinone was Q-8. The polar lipids prole of the strain showed the presence of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentied polar lipid and an unidentied phospholipid. In addition, the digital DNA-DNA hybridization values between strain NEAU-DD11 T and M. phosphatilytica 12-OD1 T and M. putida 6NM-7 T were 45.4% and 35.6%, respectively, which are lower than the accepted threshold value of 70%. The DNA G + C content of strain NEAU-DD11 T was 66.2 %. The whole genome analysis showed the strain contained carbohydrate enzymes such as glycoside hydrolase and polysaccharide lyase, which enabled the strain to had the function of degrading cellulose. On the basis of the phenotypic, genotypic and chemotaxonomic characteristics, strain NEAU-DD11 T represents a novel species of the genus Massilia, for which the name Massilia cellulosiltytica sp. nov. is proposed. The type strain is NEAU-DD11 circular, convex, smooth, and ivory-white. Good growth occurs on R2A, nutrient agar and Luria-Bertani agar. Growth occurs aerobically at 10-40 o C (optimum, 28 o C) and at pH 4.0-10.0 (optimum, 7.0). Cells grow in the presence of 0-2 % (w/v) NaCl (optimum, 0 %). Gelatin, cellulose and esculin are hydrolyzed, but starch and urea are not hydrolyzed. Arginine dihydrolase is not produced. Nitrate is reduced to nitrite. H 2 S is produced, but indole is not produced. In API ZYM, results are positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, quatic arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase and β-glucosidase, and weakly positive for α-chymotrypsin, but negative for β-glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase, α-fucosidase. In API 20NE test, D-glucose, L-arabinose, D-mannose, N-acetylglucosamine, D-maltose, potassium gluconate and malic acid are assimilated. The following substrates are not assimilated: D-mannitol, capric acid, adipic acid, citrate, phenylacetic acid. In API 50CH tests, acid is produced from D-glucose, D-fructose, D-mannose, N-acetylglucosamine, arbutin, aesculin ferric citrate, salicin, D-cellobiose, D-maltose, D-sucrose, D-trehalose and starch, and weakly produced tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate and potassium 5-ketogluconate. The major cellular fatty acids are C 16:0 , C 17:0 -cyclo and C 16:1 ω7c. The respiratory quinone is ubiquinone Q-8. The polar lipids include phosphatidylglycerol (PG), phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), an unidentied polar lipid (UL) and an unidentied phospholipid (PL). The DNA G+C content of the strain NEAU-DD11 T is 66.2 The type strain is T AB 2019141 T DSM isolated the rhizosphere soil The GenBank/EMBL/DDBJ accession for the 16S rRNA gene sequence of strain NEAU-DD11 T is This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession WSES00000000. The version this paper is version WSES00000000.1.


Introduction
The genus Massilia, belonging to the family Oxalobacteraceae, was rst described by La Scola et al. (1998) with the type species Massilia timonae isolated from blood of a patient with immunode ciency. The typical features of the genus Massilia are Gram-stain-negative, non-spore-forming, rod-shaped,  (Sun et al. 2017) and a human clinical specimen (Kämpfer et al. 2008(Kämpfer et al. , 2012. Members of the genus Massilia are widely distributed and have strong adaptability to the environment, and possess potential important application values, such as soil remediation, enzyme production and other metabolites. Massilia putida (Guang et al. 2016) isolated from ore can produce dimethyl disul de, which has potential application value for controlling soil borne diseases. Massilia chloroacetimidivorans (Lee et al. 2017) was isolated from farmland soil, which can degrade chloracetamide. Massilia tieshanensis was isolated from metal ore soil by Du et al. (2012), which was sensitive to As 3+ Cu 2+ Sb 3+ Zn 2+ Ni 2+ and Cd 2+ .
Massilia phosphatilytica 12-OD1 T was isolated from a long-term fertilized soil, which was rstly reported that it had the ability of dissolving phosphorus (Zheng et al. 2017). During an investigation of the diversity and community structure and function of microbes in the rhizosphere soil of rice, a bacterial strain NEAU-DD11 T was isolated, which could degrade cellulose. In this study, the taxonomic location of the novel strain was determined by a polyphasic approach, which is closely and phylogenetically related to the genus Massilia.

Materials And Methods
Isolation, maintenance and cultural conditions of strain Strain NEAU-DD11 T was isolated from rhizosphere soil of rice (Oryza sativa L.) collected from Northeast Agriculture University, Harbin, Heilongjiang province, North-east China (45°44′N, 126°43′E). In order to obtain the bacteria, two grams of rice rhizosphere soil were added to a 100 ml conical ask containing 18 ml of sterile distilled water and stirred in a rotating shaker at 200 rpm for 30 min and continuously diluted to a nal dilution of 10 − 3 , 10 − 4 and 10 − 5 . 200 µl of each soil suspension was plated on Reasoner's 2A (R2A) medium (0.5g yeast extract, 0.5g peptone, 0.5g casamino acid, 0.5g glucose, 0.5g soluble starch, 0.3g K 2 HPO 4 , 0.05g MgSO 4 ·7H 2 O, 0.3g sodium pyruvate, 20g agar, 1l sterile distilled water, pH 7.2) supplemented with cycloheximide (50 mg l − 1 ) and chloramphenicol (0.1 g l − 1 ). The strain NEAU-DD11 T was isolated and puri ed on R2A agar medium. After 3 days of aerobic incubation at 28 o C, the cultures were preserved as a suspension in R2A broth with glycerol (40 %, w/v) and stored at -80 o C. The reference strains Massilia phosphatilytica 12-OD1 T was obtained from the China Center for type Culture Collection (CCTCC) and Massilia putida 6NM-7 T was obtained from Guangdong Microbial Culture Center (GDMCC). These strains were cultured under the same conditions for comparative analysis. NEAU-DD11 T was tested on R2A agar, Luria-Bertani agar (5 g yeast extract, 10 g tryptone, 10 g NaCl, 20 g agar and 1 l distilled water) and nutrient agar (5 g peptone, 3 g meat extracts, 15 g agar, 1 l distilled water, pH 7.2-7.4) at 28 o C for 3 days. Color determination was done with color chips from the ISCC-NBS color charts (Kelly 1964). Bacterial growth at 4, 10, 15, 20, 25, 28, 37, 40 and 45 o C was assessed after 3 days of incubation on R2A agar. The pH range for growth was determined after 3 days of incubation at 28 o C in R2A broth adjusted to pH 1.0-12.0 at intervals of 1.0 pH units that was buffered with 0. . Tolerance to NaCl was tested in R2A broth supplemented with 0-4.0 % NaCl (w/v) with an interval of 0.5 % (w/v) after 3 days of incubation. The Gram reaction was determined by using the Hucker staining method (Lányi 1987). Degradation of cellulose and starch was determined using the methods of Smibert and Krieg (Smibert et al. 1994) after 3 days of incubation.

Morphological, cultural and physiological Characteristics
Oxidase activity was tested using an oxidase reagent (bioMérieux) according to the manufacturer's instructions. Hydrolysis of Tweens (20, 40 and 80) was tested by using the methods of Lányi (1987).
Other physiological and biochemical characteristics were examined with the API ZYM, API 20NE and API 50CH systems (bioMérieux). The API 20NE and API 50CH tests were read after 72 h incubation at 28 o C.
The API ZYM tests were read after 4 h incubation at 37 o C. Cells of strain NEAU-DD11 T grown on R2A agar at 28 o C for 2 days were used for API tests. Congo red dyeing plate (Teather et al. 1982) was used for cellulose degradation test.

Chemotaxonomic characterisation
For analysis of fatty acids, strain NEAU-DD11 T and its reference strains were grown in R2A broth at 28 o C for 3 days. Fatty acid methyl esters were extracted from the biomass according the modi ed method (cells were harvested by centrifugation and freeze-dried) as described by Gao et al. (2014) and analyzed by GC-MS using the method of Xiang et al. (2011) and identi ed with the NIST 14 database. The respiratory quinone was extracted from freeze-dried biomass and puri ed according to Collins (1985) and analyzed by using reversed-phase HPLC according to the method described by Zhao et al. (2020). The polar lipids were examined by two-dimensional TLC and identi ed using the method of Minnikin et al. (1984). Polar lipids were analyzed by using chloroform/methanol/water (65:25:4) in the rst dimension, followed by chloroform/acetic acid/methanol/water (80:18:12:5) in the second dimension.
DNA preparation, ampli cation and determination of 16S rRNA gene sequences and phylogenetic relationships Extraction of chromosomal DNA and PCR ampli cation of the 16S rRNA gene sequence were carried out using the methods of Kim et al. (2014) PCR ampli cation was carried out using the universal primers 27F and 1492R (Embley 1991). The PCR product was puri ed and cloned into the vector pMD19-T (Takara) and sequenced using an Applied Biosystems DNA sequencer (model 3730XL). Almost full-length 16S rRNA gene sequence of strain NEAU-DD11 T was obtained and compared with type strains available in the EzBioCloud server (https://www.ezbiocloud.net/identify) (Yoon et al. 2017a) and retrieved using NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi;), and then submitted to the GenBank database.
Phylogenetic trees were constructed based on the 16S rRNA gene sequences of strain NEAU-DD11 T and related reference species. Sequences were multiply aligned in Molecular Evolutionary Genetics Analysis (MEGA) using the Clustal W algorithm and trimmed manually where necessary. Phylogenetic trees were constructed with neighbour-joining (NJ) (Saitou et al. 1987) and maximum-likelihood (ML) (Felsenstein 1981) algorithms using MEGA software version X (Kumar et al. 2018). The stability of the clades of the phylogenetic trees was assessed using the bootstrap method with 1000 replications (Felsenstein 1985). A distance matrix was generated using Kimura's two-parameter model (Kimura 1980). 16S rRNA gene sequence similarities between strains were calculated on the basis of pairwise alignment using the EzBioCloud server (Yoon et al. 2017a). Phylogenetic analysis was performed as described above. Wholegenome phylogeny was generated using TYGS server (http://tygs.dsmz.de) (Meier-Kolthoff et al. 2019).
The strain NEAU-DD11 T and all the species with validly published names of the genus Massilia were included in the phylogenetic trees. Burkholderia metallica LMG 24068 T was taken as an outgroup.
For draft genome sequencing and assembly, the genomic DNA of strain NEAU-DD11 T was extracted with the SDS method (Nikodinovic et al. 2003). The harvested DNA was detected by the agarose gel electrophoresis and quanti ed by Qubit. Whole-genome sequencing was performed on the Illumina NovaSeq PE150 platform. A-tailed, ligated to paired-end adaptors and PCR ampli ed 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 ltered using the readfq (Version 10) remove reads with less than a certain percentage of low-quality bases (mass value B 38 or default is 40 bp), a certain percentage of reads with N bases (default is 10 bp), overlap exceeds a certain threshold (default is 15 bp) and the possibility reads originating from the host. All good quality paired reads were assembled using the SOAP denovo (Li et al. 2008(Li et al. , 2010. The results of morphological, physiological and biochemical characteristics that differentiated strain NEAU-DD11 T from closely related species, M. phosphatilytica 12-OD1 T and M. putida 6NM-7 T , are listed in Table 1. In API ZYM test, it was positive for lipase, leucine arylamidase, valine arylamidase, quatic arylamidase, trypsin, acid phosphatase, α-galactosidase and β-galactosidase, weakly positive for αchymotrypsin, and negative for N-acetyl-β-glucosaminidase and α-mannosidase, which were different from M. phosphatilytica 12-OD1 T . In API 20NE test, adipic acid, citrate and phenylacetic acid are assimilated except for malic acid, these also showed a distinct difference from the reference strain M. phosphatilytica 12-OD1 T . The strain could utilize N-acetyl-glucosamine, arbutin, salicin and D-brodiose, while its reference strains M. phosphatilytica 12-OD1 T and M. putida 6NM-7 T could not. These phenotypic characteristics could clearly distinguish strain NEAU-DD11 T from its closely related phylogenetic neighbours of the genus Massilia. Cellulose degradation test result showed that the strain NEAU-DD11 T had degradation ability and the degradation diameter reached 32.1 mm (Fig. S1).

Chemotaxonomic characteristics
Cellular fatty acid pro les of strains NEAU-DD11 T , M. phosphatilytica 12-OD1 T and M. putida 6NM-7 T are shown in Table S1. The fatty acid pro le of strain NEAU-DD11 T was similar to those of members of genus Massilia, with minor differences from its reference strains of some fatty acids. Strain NEAU-DD11 T contained C 16:0 (41.6 %), C 17:0 -cyclo (40.3 %) and C 16:1 ω7c (10.8 %) as the major components, which were the same with those of reference strains; but the strain did not contain C 12:0 and C 15:0 , while the reference strain M. phosphatilytica 12-OD1 T contained C 15:0 , and the reference strain M. putida 6NM-7 T contained C 12:0 . The respiratory quinone was Q-8. The polar lipids included phosphatidylglycerol (PG), phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), an unidenti ed polar lipid (UL) and an unidenti ed phospholipid (PL) (Fig. S2), which was distinct differences from its reference strains (Table   1). All these chemotaxonomic data showed that strain NEAU-DD11 T should be assigned to the genus Massilia. Table 1 Differential characteristics of strain NEAU-DD11 T , M. phosphatilytica 12-OD1 T (Fig. 2). This similar phylogenetic relationship was also observed in the maximumlikelihood (ML) tree with a bootstrap value of 54 % (Fig. S3). The phylogenetic trees generated with the NJ and ML methods showed that strain NEAU-DD11 T was grouped with members of the genus Massilia.
The draft genome of strain NEAU-DD11 T consisted of 7 341 311 bp and 30 contigs with an N50 contig length of 705 160 bp, a DNA G+C content of 66.2 % and a coverage of 130×. It was deposited in GenBank under the accession number WSES00000000. The NCBI Prokaryotic Genome Annotation Pipeline (PGAP) revealed that the genome contained four copies of the 5S rRNA genes, ve copies of the 16S rRNA genes, three copies of the 23S rRNA genes, 76 tRNA genes and four copies of noncoding RNA genes. In the draft genome of strain NEAU-DD11 T , the 16S rRNA gene sequence (MN784464) determined by PCR method was found and con rmed. Detailed genomic information is presented in Table S2. In whole genome phylogeny (Fig. 3), the strain NEAU-DD11 T formed a cluster with M. phosphatilytica 12-OD1 T and M. putida 6NM-7 T , the closest relative based on 16S rRNA gene identity. It was also supported by the highest pairwise averagenucleotide identity (ANI) value, indicating that they are closely related in many ways. These results support the conclusion that strain NEAU-DD11 T represents a novel species of the genus Massilia.
Gene function annotation can be found in the GO function classi cation diagram (Fig. S4). The genome has been identi ed as free of contamination. The genome of strain NEAU-DD11 T contained a total of 5935 genes and 5412 genes were annotated and assigned to putative functions based on the KEGG database. The result showed that more than 2489 genes of strain NEAU-DD11 T were annotated into metabolism associated pathways. The detailed distribution of genes in the KEGG functions of metabolism is shown in Fig. S5  Asterisks (*) indicate branches also recovered in the maximum-likelihood tree. Bar, 0.01 nucleotide substitutions per site.