Phenotypic characteristics
Morphological observation of strain NEAU-DD11T grown on R2A medium at 28 oC for 3 days were conducted by light microscopy and transmission electron microscopy. It revealed that it has the typical characteristics of the members of the genus Massilia. Cells of strain NEAU-DD11T were observed to be rod-shaped (1.1-1.2 µm long and 0.3-0.4 µm in diameter) and motile by peritrichous flagella (Fig. 1). The strain NEAU-DD11T grew well on R2A agar, nutrient agar and Luria-Bertani agar. Colonies were circular, convex, smooth and ivory-white on R2A agar plates. The strain NEAU-DD11T was found to grow at 10-40 oC, pH 4.0-10.0 and 0-2 % (w/v) NaCl. Optimal growth occurred at 28 oC and pH 7.0 without NaCl. The Cells are Gram-stain negative. The strain was oxidase positive, but Tweens (20, 40 and 80) were negative. The results of morphological, physiological and biochemical characteristics that differentiated strain NEAU-DD11T from closely related species, M. phosphatilytica 12-OD1T and M. putida 6NM-7T, 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 α-chymotrypsin, and negative for N-acetyl-β-glucosaminidase and α-mannosidase, which were different from M. phosphatilytica 12-OD1T. 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-OD1T. The strain could utilize N-acetyl-glucosamine, arbutin, salicin and D-fibrodiose, while its reference strains M. phosphatilytica 12-OD1T and M. putida 6NM-7T could not. These phenotypic characteristics could clearly distinguish strain NEAU-DD11T from its closely related phylogenetic neighbours of the genus Massilia. Cellulose degradation test result showed that the strain NEAU-DD11T had degradation ability and the degradation diameter reached 32.1 mm (Fig. S1).
Chemotaxonomic characteristics
Cellular fatty acid profiles of strains NEAU-DD11T, M. phosphatilytica 12-OD1T and M. putida 6NM-7T are shown in Table S1. The fatty acid profile of strain NEAU-DD11T was similar to those of members of genus Massilia, with minor differences from its reference strains of some fatty acids. Strain NEAU-DD11T contained C16:0 (41.6 %), C17:0-cyclo (40.3 %) and C16:1ω7c (10.8 %) as the major components, which were the same with those of reference strains; but the strain did not contain C12:0 and C15:0, while the reference strain M. phosphatilytica 12-OD1T contained C15:0, and the reference strain M. putida 6NM-7T contained C12:0. The respiratory quinone was Q-8. The polar lipids included phosphatidylglycerol (PG), phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), an unidentified polar lipid (UL) and an unidentified phospholipid (PL) (Fig. S2), which was distinct differences from its reference strains (Table 1). All these chemotaxonomic data showed that strain NEAU-DD11T should be assigned to the genus Massilia.
Table 1
Differential characteristics of strain NEAU-DD11T, M. phosphatilytica 12-OD1T and M. putida 6NM-7T.
Strains: 1, NEAU-DD11T; 2, M. phosphatilytica 12-OD1T; 3, M. putida 6NM-7T. All data were determined in the present study except where marked. +, positive; -, negative; w, weakly positive.
|
Characteristic
|
1
|
2
|
3
|
Colonies on R2A agar
|
|
|
|
Color
|
Ivory-white
|
Light-yellow
|
Grey-white
|
Smooth
|
+
|
-
|
-
|
NaCl (%, w/v)
|
0-2
|
0-0.5
|
0-0.5
|
Temperature (oC)
|
10-40
|
4-37
|
25-37
|
pH
|
4-10
|
5-8
|
6-8
|
API ZYM:
|
|
|
|
Lipase C14
|
+
|
-
|
+
|
Leucine arylamidase
|
+
|
-
|
+
|
Valine arylamidase
|
+
|
-
|
+
|
Cystine arylamidase
|
+
|
-
|
+
|
Trypsin
|
+
|
-
|
-
|
α-chymotrypsin
|
w
|
+
|
+
|
Acid phosphatase
|
+
|
-
|
+
|
α-galactosidase
|
+
|
-
|
+
|
β-galactosidase
|
+
|
-
|
+
|
N-acetyl-β-glucosaminidase
|
-
|
+
|
+
|
α-mannosidase
|
-
|
w
|
-
|
API 20NE:
|
|
|
|
Nitrate reduction
|
+
|
+
|
-
|
Aesculin hydrolysis
|
w
|
-
|
-
|
β-galactosidase
|
-
|
+
|
-
|
Adipic acid
|
-
|
+
|
-
|
Malic acid
|
+
|
+
|
-
|
Citrate
|
-
|
+
|
-
|
Phenylacetic acid
|
-
|
+
|
-
|
API 50CH:
|
|
|
|
D-Arabinose
|
-
|
+
|
-
|
L-Arabinose
|
-
|
+
|
+
|
D-ribose
|
w
|
-
|
-
|
D-xylose
|
-
|
+
|
-
|
D-galactose
|
-
|
+
|
+
|
D-glucose
|
+
|
-
|
+
|
D-fructose
|
+
|
+
|
w
|
N-Acetylglucosamine
|
+
|
-
|
-
|
Amygdalin
|
-
|
+
|
-
|
Arbutin
|
+
|
-
|
-
|
Salicin
|
+
|
-
|
-
|
Cellobiose
|
+
|
+
|
-
|
Raffinose
|
-
|
+
|
-
|
Glycogen
|
w
|
+
|
-
|
Gentiobiose
|
-
|
-
|
w
|
D-Tagatose
|
-
|
-
|
w
|
D-Fucose
|
-
|
+
|
-
|
L-Fucose
|
-
|
+
|
-
|
Potassium gluconate
|
-
|
+
|
+
|
Potassium 2-ketogluconate
|
-
|
+
|
-
|
Polar lipids
|
DPG, PE, PG, UL, PL
|
DPG, PE, PG, UL
|
DPG, PE, PG, UL, PL, APL
|
Major fatty acids
|
C16:0, C16:1 ω7c, C17:0-cyclo
|
C16:0, C16:1 ω7c, C17:0-cyclo
|
C16:0, C16:1 ω7c, C17:0-cyclo
|
DNA G+C content (mol %)
|
66.2
|
67.7a
|
66.8±0.6b
|
*Data taken from: a, Zheng et al. (2017); b, Guang et al. (2016)
|
Molecular characteristics
An almost-complete 16S rRNA gene sequence of strain NEAU-DD11T (1521 bp) was subjected to comparative analysis. Sequence analysis of the 16S rRNA gene showed that strain NEAU-DD11T is closely related to M. phosphatilytica 12-OD1T (98.46 %) and M. putida 6NM-7T (98.41 %). Furthermore, the neighbour-joining (NJ) phylogenetic tree based on 16S rRNA gene sequences showed that strain NEAU-DD11T formed a stable cluster with M. phosphatilytica 12-OD1T and M. putida 6NM-7T 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-DD11T was grouped with members of the genus Massilia.
The draft genome of strain NEAU-DD11T 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, 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-DD11T, the 16S rRNA gene sequence (MN784464) determined by PCR method was found and confirmed. Detailed genomic information is presented in Table S2. The digital DNA-DNA hybridization values between strain NEAU-DD11T and M. phosphatilytica 12-OD1T and M. 6NM-7T 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-DD11T 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 et al. 2009; Chun et al 2014). In whole genome phylogeny (Fig. 3), the strain NEAU-DD11T formed a cluster with M. phosphatilytica 12-OD1T and M. putida 6NM-7T, 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-DD11T represents a novel species of the genus Massilia.
Gene function annotation can be found in the GO function classification diagram (Fig. S4). The genome has been identified as free of contamination. The genome of strain NEAU-DD11T 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-DD11T were annotated into metabolism associated pathways. The detailed distribution of genes in the KEGG functions of metabolism is shown in Fig. S5. Moreover, the genome of strain NEAU-DD11T contained 47 carbohydrate-binding modules, 26 carbohydrate esterases, 207 glycoside hydrolases, 94 glycosyl transferases, 20 polysaccharide lyases, 3 auxiliary activities were annotated based on Carbohydrate-Active EnZymes database (CAZy) (Cantarel et al. 2009). These carbohydrate enzymes catalyze carbohydrate degradation, modification, and biosynthesis, and have important application value. There were many types of glycoside hydrolases, which can hydrolyze polysaccharides such as cellulose, starch, xylan and mannan (Suleiman et al. 2020). Furthermore, the strain comprised gene encoding of polysaccharide lyases that degrades cellulose into small molecules, thus playing a key role in agricultural waste utilization (Kikuchi et al. 2020). AntiSMASH version 2.0.2 (Medema et al. 2011) was used to investigate the genome contained secondary metabolites gene clusters including Terpene (3 clusters), Bacteriocin (2 clusters), Siderophore (1 cluster). For pathogenicity and drug resistance analyses, the genome of strain NEAU-DD11T was compared with the Virulence Factors of Pathogenic Bacteria Database (VFDB) (Chen et al. 2012) and Antibiotic Resistance Genes Database (Liu et al. 2009) (ARDB). The results showed that 486 virulence factors and 27 antibiotic resistance genes were annotated.
Based on the distinct phenotypic, biochemical, chemotaxonomic and phylogenetic data mentioned above, the strain NEAU-DD11T represents a novel species of the genus Massilia, for which the name Massiliacellulosiltytica sp. nov. is proposed.
Description of Massilia cellulosiltytica sp. nov.
Massilia cellulosiltytica (cel.lu.lo.si.ly’ti.ca. N.L. neut. n. cellulosum cellulose; N.L. fem. adj. lytica able to loose, able to dissolve; from Gr. fem. adj. lytikê able to loose, able to dissolve; N.L. fem. adj. cellulosilytica dissolving cellulose)
Cells are Gram-stain negative, aerobic, non-spore-forming, rod-shaped, and 0.3-0.4 × 1.1-1.2 μm in size after cultivation for 2 days at 28oC on R2A agar. Colonies on R2A agar are circular, convex, smooth, and ivory-white. Good growth occurs on R2A, nutrient agar and Luria-Bertani agar. Growth occurs aerobically at 10-40 oC (optimum, 28 oC) 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. H2S 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 from D-ribose and glycogen. Acid is not produced from glycerol, erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, D-adonitol, methyl β-D-xylopyranoside, D-galactose, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl α-D-mannopyranoside, methyl α-D-glucopyranoside, amygdalin, D-lactose, D-melibiose, inulin, D-melezitose, D-raffinose, xylitol, D-gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-ketogluconate and potassium 5-ketogluconate. The major cellular fatty acids are C16:0, C17:0-cyclo and C16:1ω7c. The respiratory quinone is ubiquinone Q-8. The polar lipids include phosphatidylglycerol (PG), phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), an unidentified polar lipid (UL) and an unidentified phospholipid (PL). The DNA G+C content of the strain NEAU-DD11T is 66.2 %.
The type strain is NEAU-DD11T (=CCTCC AB 2019141T = DSM 109721T), 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-DD11T 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.