Introduction

The genus Massilia was proposed by La Scola et al. in 1998 based on the species Massilia timonae which was isolated from blood of an immunocompromised patient with cerebellar lesions [1], and was emended later by Kämpfer et al. in 2011 [2] and Singh et al. in 2015 [3], respectively. At the time of writing, over 40 novel species within this genus were validly published (https://lpsn.dsmz.de/genus/massilia). The species within this genus were isolated from blood, ice, rock surface, soil, air and so on [1, 3,4,5,6,7,8,9]. Cells are Gram-strain-negative and rod-shaped. Q-8 is the predominant respiratory quinone and the major cellular fatty acids consist of C16:0 and C16:1 ω7c. The major polar lipids are phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The G + C contents range from 62.4 to 68.9 mol% [2, 10]. During a microbial resource survey of rivers located in the tropical and subtropical area in China, strain FT127WT was isolated from a stream. The present study determined the taxonomic status of the isolated strain.

Materials and Methods

Isolation of the Bacterial Strain and Culture Conditions

Strain FT127WT was isolated from a stream named Fentian (23° 49′ 21″ N, 113° 44′ 08″ E). The water temperature was 20.7 °C, pH was 7.6 and water depth was about 20 cm at the sampling time. Water sample was collected using the sterile bottle and stored under 4 °C. During isolation, 200 μL water sample was spread onto the agar plate containing 0.5 g yeast extract l−1, 0.5 g tryptone l−1, 0.5 g KCl l−1, 0.5 g NaCl l−1 and 20 g agar l−1 with an adjusted pH of 7.0. After incubation under 20 °C for 3 days, several single colonies were transferred onto new agar plates separately and this step was repeated for several times until the strains were purified. Strains Massilia buxea KCTC 52429T (purchased from KCTC), Massilia plicata KCTC 12344T (purchased from KCTC) and Massilia armeniaca ZMN-3 T (kindly donated by Dr. Hai-Dong Huang) were taken as reference strains in this study. All strains were routinely maintained, grown on R2A agar plates and preserved as glycerol stocks with a final concentration of 30% at − 80 °C.

Morphological, Physiological and Biochemical Characteristics

Cell morphology was observed under a light microscope (BH-2, Olympus) and a transmission electron microscope (H-9500E, Hitachi) with cells grown in R2A broth for 24 h at 24 °C. Gram staining was performed as previously described [11]. Motility was observed by stab inoculation in R2A medium with 5 g agar l−1. Growth in different NaCl concentrations (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0%, w/v) was examined in R2A broth at 24 °C. Growth at pH (4.0–10.5 at 0.5 unit intervals) was tested in R2A broth at 24 °C. pH values were adjusted using the buffer system as described by Lu et al. [12]. Growth at different temperatures (4, 10, 15, 20, 24, 30, 34, 37, 40 and 45 °C) was also assessed in R2A broth without NaCl. Catalase activity was tested by observing bubble production in 3% (v/v) H2O2. Oxidase activity was tested by adding a drop of 1% (w/v) tetramethyl-β-phenylenediamine to the strain and observing the color change. Anaerobic growth was tested by incubation in an anaerobic chamber at 24 °C for 3 days on R2A agar plates. The hydrolysis of starch, gelatin, casein, Tweens (20, 40, 60 and 80) and aesculin, urease, phenylalanine deaminase, indole production, H2S production, nitrate reduction, Voges–Proskauer and methyl red were also tested following the methods proposed by Dong and Cai [11]. API ZYM strip was used to detect enzyme activities. Single carbon-source assimilation tests were performed using a basal medium [13] containing the following (%, w/v): NaCl, 0.05; KCl, 0.02; MgSO4·7H2O, 0.02; KNO3, 0.05; (NH4)2HPO4, 0.05; KH2PO4, 0.05. Filter-sterilized saccharide, alcohol, organic acid or amino acid was added to this medium at the final concentrations of 0.2%, 0.2%, 0.1% and 0.1% (w/v), respectively. When amino acids were used as substrates, the basal medium contained neither KNO3 nor (NH4)2HPO4. After 36 h of cultivation, the optical density at 600 nm wavelength was measured with a UV spectrophotometer (UV-1200, MAPADA) to determine growth in the inoculated test-tube comparing with the control. The organic compounds used as the sole source of carbon and energy or carbon, nitrogen and energy were the following: maltose monohydrate, d-lactose monohydrate, d-glucose, sucrose, d-fructose, d-trehalose dihydrate, l-arabinose, starch, glycerol, mannitol, d-sorbitol, glycine, l-serine, l-valine, l-proline, l-aspartic acid, l-glutamic acid, sodium oxalate, sodium formate dihydrate, sodium pyruvate, disodium fumarate and sodium acetate anhydrous. Antibiotic-sensitivity tests were conducted using filter-paper discs containing the following 30 compounds (μg disc−1, unless otherwise stated): penicillin (10 U disc−1), oxacillin (1), ampicillin (10), carbenicillin (100), piperacillin (100), cephalexin (30), cefamezin (30), cefradine (30), cefuroxime (30), ceftazidime (30), ceftriaxone (30), cefoperazone (75), amikacin (30), gentamicin (10), kanamycin (30), neomycin (30), tetracycline (30), doxycycline (30), minocycline (30), erythromycin (15), norfloxacin (10), midecamycinum (30), ofloxacin (5), ciprofloxacin (5), vancomycin (30), polymyxin B (300 IU disc−1), compound sulfamethoxazole (1.25), furazolidone (300), chloramphenicol (30) and clindamycin (2). The discs were placed onto R2A agar plates spread with the strains and then incubated at 24 °C for 48 h. The inhibition zones were assessed as previously described [14].

Chemotaxonomic Analysis

Strain FT127WT and three reference strains were cultivated in R2A broth at pH 7.0 and 24 °C to exponential phases and the cells were harvested for chemotaxonomic comparisons. Cellular fatty acids were saponified, methylated and extracted in accordance with the methods described by Kuykendall et al. [15]. The fatty acid methyl ester mixtures were separated using the Sherlock Microbial Identification System and identified using the Microbial Identification software package [16]. Respiratory quinone analysis was performed by HPLC [17]. Polar lipids were extracted with chloroform/methanol (1:2, v/v) and identified by 2D TLC followed by spraying with the appropriate detection reagent [18].

Molecular Analysis

Genomic DNA of FT127WT was isolated from the overnight cell suspension culture using the genomic DNA Purification Kit. The 16S rRNA gene sequence was amplified as previously described by Lane [19] using primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′). The PCR product was purified, ligated to the vector using a commercial vector kit (Tsingke Biology & Technology Company, Beijing, China) and then transferred into Escherichia coli DH5α competent cells. The plasmid DNA was extracted and pair-end sequenced using primers M13F (5′-TGTAAAACGACGGCCAGT-3′) and M13R (5′-CAGGAAACAGCTATGACC-3′) from a biotechnology company (Tsingke Biology & Technology Company, Beijing, China). Then, the sequence was compared with the available sequences in the EzBioCloud database (https://www.ezbiocloud.net) using identify analysis [20] and the NCBI database using BLAST search [21]. The 16S rRNA gene sequence of strain FT127WT was aligned with the related type strains using the CLUSTAL_X program [22]. Evolutionary distances were calculated with the Kimura’s two-parameter model [23]. The phylogenetic trees were built using the neighbor-joining [24], maximum-parsimony [25] and maximum-likelihood methods [26] with bootstrap values based on 1000 replications in MEGA 7.0 [27]. The GenBank accession number for the 16S rRNA gene sequence of strain FT127WT was MN865813.

The genome of strain FT127WT and M. buxea KCTC 52429T were sequenced by applying the Illumina NovaSeq 6000 platform (Beijing Annoroad Gene Technology Co., Ltd). SPAdes software (https://cab.spbu.ru/software/spades/) was used to do genome assembly with multiple-Kmer parameters [28]. The G + C contents of the genomic DNA were calculated from the sequenced genome (https://www.ezbiocloud.net/tools/ani). Automated genome annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [29]. The functions of coding sequences were annotated with online tools eggNOG-Mapper [30] and BlastKOALA [31], respectively. Average nucleotide identity (ANI) analyses with whole genome sequences were estimated using ChunLab's online calculator (https://www.ezbiocloud.net/tools/ani) [32]. Digital DNA–DNA hybridization (DDH) calculations for whole genome sequences were performed using DSMZ’s online service (https://ggdc.dsmz.de/) [33]. The phylogenomic tree was built using UBCG pipeline [34]. The GenBank/EMBL/DDBJ accession numbers for the draft genome sequences of strains FT127WT and M. buxea KCTC 52429T were WWCU00000000 and WNKZ00000000, respectively.

Results and Discussion

Morphological, Physiological and Biochemical Characteristics

Cells of strain FT127WT were Gram-stain-negative, rod-shaped and motile with several flagella (Supplementary Fig. 1). All the tested strain were positive for nitrate reduction except strain M. plicata KCTC 12344T. Only strain FT127WT could not hydrolyse starch. Only strain FT127WT could utilize d-lactose monohydrate as single carbohydrate for growth, but not d-trehalose dihydrate. All the tested strains were sensitive to carbenicillin, piperacillin, cefamezin, cefuroxime, cefoperazone, amikacin, gentamicin, kanamycin, neomycin, tetracycline, doxycycline, minocycline, norfloxacin, ofloxacin, ciprofloxacin, vancomycin and compound sulfamethoxazole, but resistant to oxacillin, furazolidone, chloramphenicol and clindamycin. Detailed results of the morphological, physiological and biochemical characteristics of strain FT127WT and the reference strains are given in Table 1.

Table 1 The different characteristics of strain FT127WT and three reference strains
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Chemotaxonomic Analysis

Strain FT127WT contained Q-8 as sole respiratory quinone. It was similar to the respiratory quinone profiles of other members within genus Massilia [2,3,4,5,6,7,8,9,10]. The cellular fatty acid profiles were uniform among strain FT127WT and the reference strains. All the strains contained C16:1 ω7c, C16:0 and C12:0 as major fatty acids (Table 2), that were similar to the fatty acids profiles of other members within genus Massilia [5,6,7]. Only strain FT127WT did not contain C14:0 2OH. The polar lipids of strain FT127WT consisted of phosphatidylethanolamine, phosphatidylglycerol and one unidentified phospholipid (Supplementary Fig. S2). Diphosphatidylglycerol as a major polar lipid of genus Massilia was not detected in the polar lipids profile of strain FT127WT, that was different from the polar lipid profiles of reference strains [5, 6].

Table 2 The compositions of cellular fatty acid contents (%) of strain FT127WT and three reference strains
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Molecular Analysis

The almost complete 16S rRNA gene sequence of strain FT127WT (1495 bp in length) was determined. Comparisons of the sequence with the corresponding 16S rRNA gene sequences in the EzBioCloud and GenBank databases clearly demonstrated that strain FT127WT was a member of genus Massilia. Strain FT127WT shared 16S rRNA gene sequence similarities 98.5%, 98.3%, 98.3%, 98.1%, 97.9% and 97.6% with M. buxea A9T, M. armeniaca ZMN-3 T, M. plicata 76 T, Massilia humi THG-S6.8 T, Pseudoduganella danionis E3/2 T and M. namucuonensis 333–1-0411 T, respectively. Strain FT127WT shared 16S rRNA gene sequence similarities less than 97.6% with other related species within the family Oxalobacteraceae. The neighbor-joining tree (Supplementary Fig. S3) showed that strain FT127WT clusters closely with the species within genus Massilia. The trees reconstructed using the maximum-likelihood (Supplementary Fig. S4) and maximum-parsimony (Supplementary Fig. S5) algorithms also supported the above phylogenetic relationships.

Paired-end sequencing (2 × 150 bp) of genomic DNA fragment libraries resulted in over 7.0 × 106 quality filtered reads. Assembly of reads resulted in 177 and 359 contigs with total sequences length of 6.65 and 6.37 Mbp for strains FT127WT and M. buxea KCTC 52429 T, respectively. The G + C content was 65.3% for strain FT127WT. The sequencing coverages were about 170.3 and 238-fold for strains FT127WT and M. buxea KCTC 52429 T, respectively. The PGAP predicted 5,826 and 5,461 coding DNA sequences for strains FT127WT and M. buxea KCTC 52429 T, respectively. The functional annotations of genomes showed that strains FT127WT and M. buxea KCTC 52429 T all contain genes encoding nitrate reductase (NarGHI) and nitric oxide reductase (NorBC), but strain FT127WT also harbors gene encoding nitrous oxide reductase (NosZ) and the complete SOX system (SoxABCDXYZ) for thiosulfate oxidation. The calculated pairwise OrthoANIu values and digital DDH values between strain FT127WT and each of strains M. buxea KCTC 52429 T, M. armeniaca ZMN-3T, M. plicata DSM 17505 T and M. namucuonensis CGMCC 1.11014T were 78.0–83.1% and 22.1–26.6%, respectively, indicating that strain FT127WT should represent a novel species [35, 36]. The reconstructed phylogenomic tree based on concatenated 92 core genes also showed that strain FT127WT clusters closely with M. namucuonensis CGMCC 1.11014T, while M. buxea KCTC 52429 T, M. armeniaca ZMN-3T and M. plicata DSM 17505T together form an independently distinct clade (Fig. 1).

Fig. 1
figure 1

Neighbor-joining tree based on concatenated 92 core gene sequences of strains FT127WT and other related strains. Bar, 0.02 substitutions per nucleotide position. Bootstrap percentages > 50% based on 1000 replications are shown at the branch points

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Therefore, all these above phenotypic, chemotaxonomic, genotypic characteristics, OrthoANIu and digital DDH data supported that strain FT127WT should represent a novel species within genus Massilia, for which the name Massilia aquatica sp. nov. is proposed. The type strain is FT127WT.

Description of Massilia aquatica sp. nov.

Massilia aquatica (a.qua’ti.ca. L. fem. adj. aquatica Living in Water)

Cells are Gram-stain-negative, facultative anaerobic, rod-shaped, motile with several flagella, 2.7–3.6 μm in length and 1.0–1.2 μm in width. The colonies are pale, convex with a creamy circle. Growth occurs at 4–34 °C (optimum, 24 °C), pH 4.5–10.5 (optimum, 7.0–7.5) and NaCl 0–1.5% w/v (optimum, 0–0.5%, w/v). Positive for oxidase, catalase, urease and nitrate reduction, but negative for H2S production, phenylalanine deaminase, Voges-Proskauer test, methyl red test and indole production. It hydrolyses Tweens (20, 40, 60 and 80), casein and gelatin, but not aesculin or starch. It utilizes maltose monohydrate, d-lactose monohydrate, d-glucose, d-fructose, l-arabinose, starch, sodium pyruvate and sodium acetate anhydrous, but not sucrose, d-trehalose dihydrate, glycerol, mannitol, d-sorbitol, l-glutamic acid, l-aspartic acid, l-valine, glycine, l-proline, l-serine, sodium oxalate, sodium formate dihydrate or disodium fumarate. Negative for esterase (C4), esterase lipase (C8), lipase (C14), cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase, positive for alkaline phosphatase, leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and α-glucosidase. The major fatty acids are C16:1 ω7c, C16:0 and C12:0. The polar lipids includes phosphatidylethanolamine, phosphatidylglycerol and one unidentified phospholipid. The G + C content of genomic DNA is 65.3%.

Type strain FT127WT (= GDMCC 1.1690 T = KACC 21482 T) was isolated from a subtropical stream in China. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and draft genome of strain FT127WT are MN865813 and WWCU00000000, respectively.