Massilia cellulosiltytica sp. nov., a novel cellulose-degrading bacterium isolated from rhizosphere soil of rice (Oryza sativa L.) and its whole genome analysis

A bacterial strain, Gram-stain negative, rod-shaped, aerobic and cellulose-degrading, designated NEAU-DD11T, was isolated from rhizosphere soil of rice collected from Northeast Agricultural University in Harbin, Heilongjiang Province, North-east China. Base on 16S rRNA gene sequence analysis, strain NEAU-DD11T belongs to the genus Massilia and shared high sequence similarities with Massilia phosphatilytica 12-OD1T (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-DD11T formed lineage related to M. phosphatilytica 12-OD1T and M. putida 6NM-7 T. The major fatty acids of the strain were C16:0, C17:0-cyclo and C16:1ω7c. The respiratory quinone was Q-8. The polar lipids profile of the strain showed the presence of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified polar lipid and an unidentified phospholipid. In addition, the digital DNA-DNA hybridization values between strain NEAU-DD11T and M. phosphatilytica 12-OD1T 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-DD11T 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 have the function of degrading cellulose. On the basis of the phenotypic, genotypic and chemotaxonomic characteristics, we conclude that strain NEAU-DD11T represents a novel species of the genus Massilia, for which the name Massilia cellulosiltytica sp. nov. is proposed. The type strain is NEAU-DD11T (= CCTCC AB 2019141 T = DSM 109721 T).


Introduction
The genus Massilia, belonging to the family Oxalobacteraceae, was first described by La Scola et al. (1998) with the type species Massilia timonae isolated from blood of a patient with immunodeficiency. The typical features of this genus are Gram-stain-negative, non-spore-forming, rod-shaped, aerobic and motile (except for Massilia arvi THG-RS2O T , 'Massilia humi THG-S6.8 T ', Massilia glaciei B448-2 T and Massilia varians comb. CCUG 35299 T ) (Yang et al. 2019). Summed feature 3 (C 16:1 x7c and/or iso-C 15:0 2-OH), C 17:0 -cyclo and C 16:0 are the major fatty acids and Q-8 is the predominant ubiquinone. The major polar lipids are diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). The DNA G ? C contents range from 62.4 to 69.4%. At present, the genus Massilia contains a total of 55 species with validly published names (https:// www.bacterio.net/genus/massilia), including the recently described species Massilia arenosa (Rachel et al. 2020), Massilia atriviolacea (Yang et al. 2019) and Massilia aquatica (Lu et al. 2020). Members of the genus were isolated from various sources, such as soil (Chaudhary et al. 2017;Singh et al. 2015;Zu et al. 2008;Kim 2014;Zheng et al. 2017), air (Weon et al. 2008(Weon et al. , 2009, water (Gallego et al. 2006), ice core (Shen et al. 2013;Guo et al. 2016), glacier permafrost (Wang et al. 2018), rock surface (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 diverse environments, and possess potential important application values, such as soil remediation, enzyme production and production of other metabolites. Massilia putida (Guang et al. 2016) isolated from ore can produce dimethyl disulfide, 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 longterm fertilized soil, which was firstly reported to have 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 affiliation of this novel strain was determined by a polyphasic approach, which is identified as a novel species in 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 0 N, 126°43 0 E). In order to obtain the bacteria, two grams of rice rhizosphere soil were added to a 100 ml conical flask containing 18 ml of sterile distilled water and stirred in a rotating shaker at 200 rpm for 30 min and serially diluted to 10 -5 . Aliquots (200 ll) of the last three dilutions for each soil suspension was plated on Reasoner's 2A (R2A) medium (0.5 g yeast extract, 0.5 g peptone, 0.5 g casamino acid, 0.5 g glucose, 0.5 g soluble starch, 0.3 g K 2 HPO 4 , 0.05 g MgSO 4 Á7H 2 O, 0.3 g sodium pyruvate, 20 g agar, 1L sterile distilled water, pH 7.2) supplemented with cycloheximide (50 mg l -1 ) and chloramphenicol (0.1 g l -1 ). Single colonies with different colony morphologies were picked up for purification by repeatedly cross-streaking on the same medium and the taxonomic affiliations of the pure isolates were screened by analysis of 16S rRNA gene sequence as described subsequently. Isolate NEAU-DD11 T was chosen for further characterization since it presented sequence similarity lower than the suggested species threshold (98.6%) (Stackebrandt et al. 2006).The strain NEAU-DD11 T was isolated and purified on R2A agar medium. After 3 days of aerobic incubation at 28°C, the cultures were preserved as a suspension in R2A broth with glycerol (40%, w/v) and stored at -80°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.

Morphological, cultural and physiological Characteristics
Cell morphology and the presence of flagella were determined by light microscopy (ECLIPSE E200, Nikon) and transmission electron microscopy (Hitachi H-7650) using cells grown on R2A agar medium at 28°C for 72 h. The cell motility was established by observing the growth and diffusion of cells in the medium of semi-solid R2A containing 0.3% agar at 20°C incubation for 48 h (Yang et al. 2019). Growth of strain 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°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°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°C in R2A broth adjusted to pH 1.0-12.0 at intervals of 1.0 pH units that was buffered with 0.1 M citric acid/0.1 M sodium citrate; 0.1 M KH 2 PO 4 /0.1 M NaOH; 0.1 M NaHCO 3 /0.1 M Na 2 CO 3 ; 0.2 M KH 2 PO 4 /0.1 M NaOH Zhao et al. 2019). 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 (1994) after 3 days of incubation. 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°C. The API ZYM tests were read after 4 h incubation at 37°C. Cells of strain NEAU-DD11 T grown on R2A agar at 28°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°C for 3 days. Fatty acids were extracted from the biomass and methylated according the modified 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 identified with the NIST 14 database. The respiratory quinone was extracted from freeze-dried biomass and purified 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 identified using the method of Minnikin et al. (1984). Polar lipids were analyzed by using chloroform/ methanol/water (65:25:4) in the first dimension, followed by chloroform/acetic acid/methanol/water (80:18:12:5) in the second dimension.
DNA preparation, amplification and determination of 16S rRNA gene sequences and phylogenetic relationships Extraction of chromosomal DNA and PCR amplification of the 16S rRNA gene sequence were carried out using the methods of Kim and Ka (2014). PCR amplification was carried out using the universal primers 27F and 1492R (Embley 1991). The PCR product was purified 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 Gen-Bank 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 neighbourjoining (NJ) (Saitou and Nei 1987) and maximumlikelihood (ML) (Felsenstein 1981) algorithms using MEGA software version X (Sudhir 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. Whole genome sequence of strain NEAU-DD11 T was obtained as mentioned subsequently and its 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 quantified by Qubit. Wholegenome sequencing was performed on the Illumina NovaSeq PE150 platform. A-tailed, ligated to pairedend 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 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 (http://soap.genomics.org.cn/ soapdenovo.html) into a number of scaffolds. Two genomic metrics are now available to distinguish between orthologous genes of closely related prokaryotes, including the calculation of average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH). The dDDH and ANI values were determined between the genomes of strain NEAU-DD11 T and M. phosphatilytica 12-OD1 T and M. putida 6NM-7 T online at http://ggdc.dsmz.de using the Genome-to-Genome Distance Calculation (GGDC 2.0) (Meier-Kolthoff et al. 2013) and the ChunLab's online ANI Calculator (www.ezbiocloud. net/tools/ani) (Yoon et al. 2017b), respectively.

Chemotaxonomic characteristics
Cellular fatty acid profiles of strains NEAU-DD11 T , M. phosphatilytica 12-OD1 T and M. putida 6NM-7 T are shown in Table S1. The fatty acid profile 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 x7c (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 Fig. 1 Transmission electron micrograph of strain NEAU-DD11 T grown on R2A agar for 3 days at 28°C. Bar, 1 lm Leucine arylamidase ? -?
Cystine arylamidase ? -?  (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.

Molecular characteristics
An almost-complete 16S rRNA gene sequence of strain NEAU-DD11 T (1521 bp) was subjected to comparative analysis. Sequence analysis of the 16S rRNA gene showed that strain NEAU-DD11 T is closely related to M. phosphatilytica 12-OD1 T (98.46%) and M. putida 6NM-7 T (98.41%). Furthermore, the neighbour-joining (NJ) phylogenetic tree based on 16S rRNA gene sequences showed that strain NEAU-DD11 T formed a stable cluster with M. phosphatilytica 12-OD1 T and M. putida 6NM-7 T with a bootstrap value of 56% (Fig. 2). This similar phylogenetic relationship was also observed in the maximum-likelihood (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 9 . 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, five 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 confirmed. Detailed genomic information is presented in Table 2. The digital DNA-DNA hybridization values between strain NEAU-DD11 T and M. phosphatilytica 12-OD1 T and M. 6NM-7 T were 45.4% and 35.6%, respectively, which were all below the threshold value of 70% proposed for species discrimination by Wayne et al. (1987). Similarly, the low ANI values of genome sequences between strain NEAU-DD11 T and its reference strains were 91.7% and 88.4%, respectively, results well below the threshold of 95-96% used to delineate prokaryote species (Richter and Rossello-Mora 2009;Chun and Rainey 2014). 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.
Based on the distinct phenotypic, biochemical, chemotaxonomic and phylogenetic data mentioned above, the 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 T (= CCTCC AB 2019141 T = DSM 109721 T ), isolated from the rhizosphere soil of rice collected from Northeast Agricultural University in Harbin, Heilongjiang Province, China. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain NEAU-DD11 T is MN784464. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession WSES00000000. The version described in this paper is version WSES00000000.1.
Ethical approval This article does not contain any studies with human participants and/or animals performed by any of the authors. The formal consent is not required in this study.
Consent to participate This research doesn't involve in human subjects, so the informed consent to participate and consent to publish are not obtained.
Consent to publish All authors have seen a copy of the manuscript and have approved its submission.