The nearly complete 16S rRNA gene sequence of strain 3D7T (1396 bp) was determined and compared with the corresponding sequences. It shared the highest 16S rRNA gene similarity to ‘Microvirga brassicacearum’ CDVBN77T (98.3 %), followed by Microvirga subterranea DSM 14364T (96.8 %), Microvirga guangxiensis 25BT (96.5 %), and Microvirga aerophila DSM 21344T (96.5 %). The NJ analyses (Fig. 1) showed that strain 3D7T shared a branching node with ‘M. brassicacearum’ CDVBN77T, which was highly consistent with ME tree (Fig. S1) and ML tree (Fig. S2). It was clear that strain 3D7T was a member of the genus Microvirga.
Genome composition and DNA-DNA hybridisation
The draft genome sequence of strain 3D7T was 4,518,469 bp in length with 221 contigs. The coverage, N50 and DNA G + C content were 410×, 431,466 bp and 63.5 mol%. The genome had 4321 protein-coding genes and 49 RNAs (Table 1). Genomic analyses showed that strain 3D7T and ‘M. brassicacearum’ CDVBN77T yielded ANIb and dDDH values of 77.5 and 22.2 %, respectively. The ANI values between strain 3D7T and other species of the genus Microvirga are detailed in Table 1, which are all below standard criteria for classifying strains as different species (95–96 %) (Kim et al. 2014). The dDDH values between strain 3D7T and other species of the genus Microvirga are also detailed in Table 1, and they are far below the 70 % cut-off value generally recommended for species differentiation (Wayne et al. 1987). A genome-based phylogenetic tree is included in Fig. S3, which shows that strain 3D7T is affiliated to the genus Microvirga. These data confirm that strain 3D7T represents a novel species of the genus Microvirga.
Genome features and function prediction
Gene Ontology database analysis results showed that strain 3D7T has 42 different types functions. Among them, there were 14 features related to cell component, 14 features related to molecular function, 14 features related to biological process (Fig. S4). Among the 20 general COG functional categories, the detailed distribution of genes was as follows: Amino acid transport and metabolism, 497 genes; Inorganic ion transport and metabolism, 293 genes; Energy production and conversion, 204 genes; Transcription, 202 genes; Carbohydrate transport and metabolism, 200 genes. Detailed information of the COG functional categories was presented in Fig. S5. KEGG metabolic pathways were classified according to the relationship between KO (KEGG ORTHOLOGY) and Pathway. Functional annotation of genes by comparisons against the manually curated KEGG GENEs database revealed that there were 62 genes related to the biosynthesis of other secondary metabolites, 122 genes related to the biodegradation and metabolism of xenobiotics, 374 genes related to the metabolism of carbohydrates and 45 genes related to the metabolism of terpenoids and polyketides (Fig. S6). The genome annotations showed genes encoding for proteins with phosphatase activity, such as the enzymes alkaline phosphatase (EC 220.127.116.11), acid phosphatase (EC 18.104.22.168), inorganic triphosphatase (EC 22.214.171.124) and pyrophosphatase (EC 126.96.36.199). Some enzymes involved in the production of triglyceride lipases, including lysophospholipase (EC 188.8.131.52) and unidentified phospholipase were also observed. These capabilities were tentatively proven in physiological tests, with potential applications in the agriculture biotechnology, washing industry and low-temperature environment remediation.
Analysis of the genome sequence of strain 3D7T showed 132 genes encoding different CAZymes in five different classes: glycoside hydrolases (GHs), enzymes that catalyze the hydrolysis of glycosidic linkage of glucoside—27 gene counts; glycosyltransferases (GTs), involved in the formation of glycosidic bonds—47 gene counts; carbohydrate esterases (CEs), which hydrolyze carbohydrate esters—35 gene counts; auxiliary activities (AAs), redox enzymes that act in conjunction with CAZymes—20 gene counts; polysaccharide lyases (PLs), which perform non-hydrolytic cleavage of glycosidic bonds—2 gene counts and carbohydrate-binding modules(CBMs)—1 gene count (Table S1). AntiSMASH output revealed four biosynthetic gene clusters (BGCs) involved in the secondary metabolism of the bacterium. One of those clusters encodes terpene BGC, which is related to the synthesis of isoindolinomycin. Other clusters encode an arylpolyene, a hserlactone and a terpene BGCs that are not described for the production of an already known molecule. These genetic characteristics indicated that strain 3D7T may have biotechnological potential for the degradation of biomass and the pharmaceutical industry.
Strain 3D7T grew well on R2A agar and ASM agar, but grew weakly on TSA and NA. Colonies on R2A agar plate were light-pink, semi-transparent, smooth and round. Cells were Gram-stain-negative, aerobic, non-motile, rod-shaped, 1.0–2.2 µm long and 0.7–0.9 µm wide (Fig. S7). It was able to grow at 4–32 ℃ (optimum, 25–28 ℃), pH 7.0–10.0 (optimum, 7.0–7.5) and in the presence of 0–3 % (w/v) NaCl (optimum without NaCl). These characteristics markedly differentiated strain 3D7T from the first related strain ‘M. brassicacearum’ CDVBN77T. Sensitive to penicillin (10 U), ampicillin (10 µg), chloramphenicol (30 µg), tetracycline (30 µg), streptomycin (10 µg) and neomycin (30 µg), but resistant to polymyxin B (300 IU), vancomycin (30 µg) and bacitracin (0.04 U). Strain 3D7T hydrolysed Tween 20, 40, 60, and weakly hydrolysed Tween 80. It can hydrolyse aesculin, but not gelatin and tyrosine. Positive reaction for alkaline phosphatase, valine arylamidase and naphthol-AS-BI-phosphohydrolase, weakly positive for lipase (C14) and trypsin. These characteristics differentiated strain 3D7T from ‘M. brassicacearum’ CDVBN77T and other closely related reference strains. More differential characteristics between strain 3D7T and its closely related species in the genus Microvirga were given in Table 2, and the other detailed physiological and biochemical characteristics are present in the species description.
The major cellular fatty acids of strain 3D7T (> 10 %) were summed feature 8 (C18:1ω7c and/or C18:1ω6c) (36.2 %) and C19:0 cyclo ω8c (21.7 %), which was similar to that of closely related species of the genus Microvirga. Minor qualitative and quantitative differences could be used to distinguish strain 3D7T from the closest relatives of the genus Microvirga. Compared with ‘M. brassicacearum’ CDVBN77T, strain 3D7T possessed higher amounts of C16:0, summed feature 2 (C14:0 3-OH and/or iso-C16:1 I) and summed feature 3 (C16:1ω6c and/or C16:1ω7c), and lower amounts of C18:0, C18:0 3-OH, C18:1ω7c 11-methyl and feature 8 (C18:1ω7c and/or C18:1ω6c). C14:0 and C17:0 cyclo were detected in strain 3D7T, but not detected in ‘M. brassicacearum’ CDVBN77T (Table 3). The predominant respiratory quinone of strain 3D7T was Q-10, which was in good agreement with other species of the genus Microvirga. The polar lipids of strain 3D7T consisted of phosphatidylcholine and phosphatidylethanolamine as the major component, plus one unidentified aminophospholipid, two unidentified amino lipids and three unidentified lipids (Fig. S8). Strain 3D7T shared the same major polar lipids with most of the described species of the genus Microvirga.
In conclusion, all phenotypic, chemotaxonomic, phylogenetic and genomic analyses suggested that strain 3D7T should be considered to represent a novel species of the genus Microvirga, for which the name Microvirga antarctica sp. nov. is proposed.
Description of Microvirga antarctica sp. nov.
Microvirga antarctica (ant.arc'ti.ca. L. fem. adj. antarctica southern, pertaining to the Antarctica, where the type strain was isolated)
Cells are Gram-stain-negative, aerobic, non-motile and rod-shaped (0.7–0.9×1.0–2.2 µm). Growth occurs on R2A agar and ASM agar, weakly on NA and TSA. Colonies are light-pink, semi-transparent, smooth, round and smaller than 1.0 mm in diameter after 3 days at 28 ℃. Growth occurs at a range of 4–32 ℃ (optimum, 25–28 ℃) and pH 7.0–10.0 (optimum, 7.0–7.5) and in the presence of 0–3 % (w/v) NaCl (optimum without NaCl). Oxidase, catalase and nitrate reduction are positive, but glucose fermentation, arginine dihydrolase, indole production and urease are negative. Hydrolyses aesculin, Tween 20, 40 and 60, weakly hydrolyses Tween 80, but not starch. Assimilates L-arabinose, D-xylose, D-ribose and D-cellobiose, weakly assimilates D-fucose, D-glucose and D-melosanose. Positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase, weakly positive for lipase (C14), cystine arylamidase and trypsin. The major polar lipids are phosphatidylcholine and phosphatidylethanolamine. The predominant quinone is Q-10 and the major fatty acids are summed feature 8 (C18:1ω7c and/or C18:1ω6c) and C19:0 cyclo ω8c. The genomic DNA G + C content of the type strain is 63.5 mol%.
The type strain, 3D7T (= CGMCC 1.13821T = KCTC 72465T), was isolated from a soil sample collected from Deception Island, Antarctica. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 3D7T is MH561859. This Whole Genome Shotgun project of strain 3D7T has been deposited at DDBJ/ENA/GenBank under the accession number JAGEMM000000000.