16S rRNA gene sequence and phylogenetic analysis
The almost complete 16S rRNA (1,457 bp) gene sequence of strain L22T was obtained through conventional Sanger sequencing. The result of pairwise comparison of 16S rRNA gene sequences in the GenBank database of NCBI server (https://www.ncbi.nlm.nih.gov) showed that the most related strain was M. pamukkalensis JCM 30229Twith 95.9 % 16S rRNA gene sequence similarity, followed by C. albigriseusJCM 30603T (95.0 %), O. diazotrophicusDSM 102969T (94.5 %), N. lucknowenseDSM 24322T (93.8 %) and N. indicusC115T (93.8 %). The 16S rRNA gene sequence of strain L22T showed a maximum identity of 95.9 % with validly published species, which is below the 98.7 % species threshold identity but higher than the 94.5% genus threshold identity. Following the 16S rRNA-based taxonomic identity, the species rank thus seems appropriate for this strain. Besides, the phylogenetic maximum-likelihood tree based on 16S rRNA gene sequences (Fig. 1) showed than strain L22T belonged to the genus Methylobrevis. The topology of the phylogenetic tree constructed by neighbour-joining arithmetic (Fig. S1) proved the conclusion that strain L22T and M.pamukkalensisJCM 30229T formed a stable cluster which had a distinct phylogenetic lineage with other genera within the family Pleomorphomonadaceae. These overall data indicated that strain L22T was regarded as a novel species within the genus Methlobrevis.
Genome sequence analysis
The draft genome sequence of strain L22T consisted of 4,354,788 bp. The combined assembly yielded 74 contings with the largest being 618,762 bp and the coting N50 value was 249,401 bp. An average 473× coverage depth was accomplished. The DNA G+C content of strain L22T was 69.8 mol%, which was following reported value for strain M. pamukkalensis JCM 30229T (67.9 mol%) (Doronina et al. 2015). Gene prediction and annotation identified 3,927 genes, 3,866 protein-encoding genes, 51 tRNA genes, 6 rRNA genes. Two 16S rRNA gene sequences (1,167 bp and 1,473 bp) was extracted from the draft genome and were compared with the almost complete 16S rRNA (1,457 bp) gene sequence of strain L22T obtained through conventional Sanger sequencing. One shew99.9 % and the other 99.8 % gene sequence similarity. Moreover, according to annotation from KEGG database,genes assigned to functional categories are involved in the environmental information processing (318), carbohydrate metabolism (250), protein families: genetic information processing (227), protein families: signaling and cellular processing (222), genetic information processing (164) and amino acid metabolism (147). Gene katE (encoding catalase) and katE-intracellular protease were found which meant strain L22T got the ability to hydrolyze hydrogen peroxide. A complete phosphatidate cytidylyltransferase [EC:2.7.7.41] pathway and gene pmtA were proved to exist and thus Phosphatidylethanolamine (PE) can be produced and transformed into Phosphatidylcholine (PC), in accord with the result of polar lipids experiment. Results on KEGGtold that strain L22T can reduce sulfate to APS, PAPS, sulfite and finally sulfide step by step. However, the experiment showed that sodium thiosulfate could not be reduced to H2S. Phylogenetic analysis of the genomic amino acid sequences performed with the FastTree and IQTree approaches demonstrates that strain L22T formed a stable lineage within the genus Methylobrevis (Fig. 2 & Fig. 3).
The AAI value between strain L22T and M.pamukkalensis JCM 30229T was 71.8 %, significantly below the proposed cut-off for a species boundary of 85-90 % and exceed the threshold value for a genus boundary 55-60 % (Rodriguez-R and Konstantinidis 2014). The results of ANI and dDDH between strain L22T and M. pamukkalensis JCM 30229T were 78.6 % and 22.3 %, both below the thresholds (ANI: 95-96 %; dDDH: 70 %) for new species identification (Goris J et al. 2007) (Meier-Kolthoff et al. 2013). These results supported the finding that strain L22T was a novel member of the genus Methylobrevis.
Morphological, physiological and biochemical characteristics
Cells of strain L22T were Gram-stain-negative, aerobic, 0.3-0.4 μm wide and 0.9-1.1 μm long, gliding, short rods with long flagella (Fig. S2), thus made them possible to move quickly in a liquid environment. Colonies of strain L22T were milky white, convex and round could grow up to 0.5 mm in diameter after incubation on R2A agar at 30 ℃ for 3 days. Strain L22T grow at 4-37 ℃ (optimum 30 ℃), at pH 6.0-9.5 (optimum 7.5). Interestingly, though strain L22T was isolated from the fourth biggest freshwater lack in China, its tolerance to NaCl was high up to 4.5 % (w/v) (optimum 0-0.75 %). Strain L22T was susceptible to chloramphenicol (30 μg), ofloxacin (5 μg), norfloxacin (10 μg), ceftriaxone (30 μg), tetracycline (30 μg), cefotaxime (30 μg), penicillin (10 μg) and ampicillin (10 μg), resistant to clarithromycin (15 μg), rifampicin (5 μg), gentamicin (10 μg), vancomycin (30 μg), erythromycin (15 μg), streptomycin (10 µg), lincomycin (2 µg), neomycin (30 µg) and tobramycin (10 µg). The strain was negative for hydrolyzes of starch, casein, alginate, cellulose, Tweens 20, 60 and 80 but positive for Tween 40. Strain L22T show many typical characteristics of the genus Methylobrevis, such as cells are Gram-stain-negative, aerobic, rods, positive for catalase and oxidase activities, and negative for nitrate reduction(Doronina et al. 2015). However, differences remained that strain M. pamukkalensis JCM 30229T showed positive for starch and negative gelatinase activity while strain L22T was not. The other morphological, physiological, and biochemical characteristics that differentiate strain L22T from its related type strain are provided in Table 1.
Chemotaxonomic characterization
The major fatty acid (>10.0 %) of strain L22T was summed feature 8 (C16:1ω7c) (69.1 %) same as the major fatty acid extracted from its related strain strain M. pamukkalensis JCM 30229T. The polar lipids of strain L22T comprised phosphomonoester (PME), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC), one unknown glycolipid (GL) and seven unknown lipids (L). What deserved to be mentioned was that strain L22T and strain M. pamukkalensis JCM 30229T both possessed phosphomonoester (PME), phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and phosphatidylcholine (PC) as the major polar lipids (Fig. S3). Strain L22T contained unknown lipid 1 to 9, while strain 30229T did not have unknown lipid 8 and unknown lipid 9 but had unknown lipid 10 to 13.Details of cellular fatty acids and polar lipids compositions were listed in Table 2. The major ubiquinone of strain L22T was Q-10, which is accordant to the description of the family Pleomorphomonadaceae(Hrdt et al. 2020).
According to the phylogenetic analyses, differential chemotaxonomic data and other phenotypic properties, strain L22T was classified as a new member of the genus Methylobrevis.
Description of Methylobrevis albus sp. nov.
Methylobrevis albus (al’bus L. masc. adj. albus, white, describing the white aerial mycelium)
Cells are Gram-stain-negative, aerobic, gliding motility, 0.3-0.4 μm wide and 0.9-1.1 μm long rods with flagella.When grown on R2A agar, colonies were 0.5 mm in diameter after 3 days of growth at 30 ℃ at pH 7.5, milky white, convex and circular, glossy surfaces, with entire edges. Growth occurs at 4-37 ℃ (optimum 30 ℃) and pH 6.0-9.5 (optimum 7.5). Able to tolerate up to 4.5 % (w/v) NaCl (grows optimally at 0-0.75 % NaCl). Activities of oxidase and catalase are positive.Unable to reduce nitrate.The strain was negative for hydrolyzes of starch, casein, alginate, cellulose, Tweens 20, 60 and 80 but positive for Tween 40. Positive for urease, gelatinase, alkaline phosphatase, lipoidase (C4), white aramidase, valine aramidase, cystine aramidase, trypsin, naphthol-AS-BI-phosphohydrolase and α-glucosidase, but negative for β-galactosome, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan dehydrogenase, acid phosphatase, esterase lipidase (C8), lipase(C14), chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, β-glucosidase, N-acetyl-glucosinase, α-mannitase, β-fucosidase. Has the ability to produce acetyl methyl carbinol from 3-hydroxy ethyl ketone but not able to produce H2S either indole. Oxidation can be found with these carbon sources:sucrose, α-d-glucose, d-mannose, d-fructose, d-galactose, d-fucose, l-fucose, l-rhamnose, Inosine, d-sorbitol, d-mannitol, d-arabitol, myo-inositol, glycerol, d-fructose-6-PO4, l-alanine, l-serine, pectin, d-galacturonic acid, l-galactonic acid lactone, d-gluconic acid, d-glucuronic acid, glucuronamide, quinic acid, methyl pyruvate, d-lactic acid methyl ester, l-lactic acid, α-Keto-glutaric acid, d-malic acid, l-malic acid, bromo-succinic acid, β-hydroxy-d,l-butyric acid, acetoacetic acid, acetic acid and formic acid. Acids can be produced from various substrates: mannitol, l-arabinose, d-ribose, d-xylose, d-side calendula Ⅰ, d-fructose, d-sucrose, xylitol, d-lyxose, d-fucose and l-fucose. The major fatty acid is summed feature 8 (C16:1ω7c). The polar major lipids consist of phosphomonoester, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine. The sole ubiquinone is Q-10.
The type strain is L22T (=KCTC 72858T=MCCC 1H00432T), which is isolated from fresh water of Hulun Lake (117°01' 10" E, 48°30' 40" N), Inner Mongolia, PR China. The genomic DNA G+C content of the type strain is 69.8 mol %. The 16S rRNA gene sequence and the draft genome sequences are available in the NCBI GenBank database under the accession number MW195049 and JADZLT000000000.