16S rRNA gene sequence and phylogenetic analysis
The 16S rRNA gene sequence extracted from the genome assembly was 1491 bp (MG385132.2), which included the 16S rRNA gene sequence acquired from PCR and clone. Based on 16S rRNA gene sequences, R. frigidaeris JCM 32945T（97.2% sequence similarity）, P. ruber JCM 9931T (96.4% sequence similarity) and the following D. rubra JCM 30602T (95.8% sequence similarity) were the most closely related type strains to NE82T. In the neighbor-joining phylogenetic tree (Fig. 1), the strain NE82T formed a cluster with R. frigidaeris JCM 32945T, the only one species of the genus Roseicella. Phylogenetic trees were also constructed using the maximum-likelihood and maximum-parsimony algorithms (Fig. S1 and Fig. S2, available with the online Supplementary Information), which supported the result above.
The draft genome sequence of strain NE82T was 5.9 Mb in length and produced 238 contigs. Contigs varied in length from 211 bp to 414,331 bp (N50 = 166,730 bp). The G+C content of the genomic DNA of strain NE82T was 72.0 mol%. The draft genome of strain NE82T contained 5,532 genes, one 16S rRNA and 55 tRNAs annotated by the NCBI Prokaryotic Genome Annotation Pipeline. KEGG pathway annotation predicted that strain NE82T could degrade aromatic hydrocarbon, such as benzoate. Besides, the result of prediction also showed that NE82T could translate thiosulfate to sulfate via thiosulfate oxidation by SOX complex, which contributed to the sulfur cycle on Earth. Moreover, the draft genome sequence of R. frigidaeris JCM 32945T, P. ruber JCM 9931T and D. rubra JCM 30602T were also sequenced with the 100X sequencing depth, the length were 5.8 MB, 7.2 Mb and 7.8 Mb, respectively. R. frigidaeris JCM 32945T produced 87 contigs with an N50 of 187085 bp (Khan et al. 2019), P. ruber JCM 9931T produced 786 contigs (203 bp to 188,134 bp) while D. rubra JCM 30602T produced 458 contigs (202 bp to 363,070 bp). N50 of P. ruber JCM 9931T and D. rubra JCM 30602T were 22,677 bp and 79,244 bp.
The ANI between strain NE82T and R. frigidaeris JCM 32945T, P. ruber JCM 9931T, D. rubra JCM 30602T were 83.3%, 84.1% and 83.4%, respectively. The dDDH between strain NE82T and R. frigidaeris JCM 32945T, P. ruber JCM 9931T, D. rubra JCM 30602T was 27.2%, 27.6% and 26.8%, respectively. According to the proposed and generally accepted species boundary, ANI value <95% or dDDH value <70% means that the strain is a novel species (Rodriguezr and Konstantinidis 2016; Meier-Kolthoff et al. 2013), which proved strain NE82T was a novel species distinguishable from the closely related type strains.
Morphological, physiological and biochemical characterizations
Cells of strain NE82T were ellipsoidal, approximately 0.4-0.9 µm in diameter, which was found to be Gram-stain-negative, non-motile and facultatively aerobic. Colonies were red-pigmented, circular and measured about 1.0 mm in diameter on R2A agar. Growth of strain NE82T was found to occur between 15 and 42°C (optimum 28-33°C), pH 5.5-8.5 (optimum pH 7.0-7.5) and in the presence of 0-1.0% (w/v) NaCl (optimum 0%).
Strain NE82T could not grow under anaerobic conditions, with or without 1% (w/v) KNO3, after two weeks’ cultivation in an anaerobic chambre on R2A at 30°C. The test for the reduction of nitrate was positive and strain NE82T also had catalase activity, which is consistent with R. frigidaeris JCM 32945T. The hydrolysis of Tweens 20, 40, 60 were detected, but starch, casein, cellulose, alginate and Tween 80 were not hydrolysed, while the most close related strain R. frigidaeris JCM 32945T could not hydrolyse Tween 20. These results were same with P. ruber JCM 9931T , but displayed little difference with D. rubra JCM 30602T, which could hydrolysing Tween 80. Carotenoid was present in strain NE82T, R. frigidaeris JCM 32945T (Khan et al. 2019) and P. ruber JCM 9931T, while P. ruber JCM 9931T also contained Bacteriochlorophyll a (Saitoh et al. 1998). Strain NE82T was found to be susceptible to carbenicillin (100 µg), chloramphenicol (30 µg), penicillin (10 µg), tetracycline (30 µg), ampicillin (10 µg), kanamycin (30 µg), cefotaxime sodium (30 µg), erythromycin (15 µg), streptomycin (10 µg), tobramycin (10 µg), rifampicin (5 µg), gentamicin (10 µg), but resistant to norfloxacin (30 µg), vancomycin (30 µg), lincomycin (2 µg), clindamycin (30 µg). Despite strain NE82T showed many common traits with R. frigidaeris JCM 32945T, it could be distinguished from this strain by a number of biochemical characteristics, such as the negative reaction of oxidase reaction, valine arylamidase, gelatinase and Voges–Proskauer reaction, the positive utilization of urease and citrate. The complete morphological, physiological and biochemical analyses are summarised in Table 1.
The predominant cellular fatty acids of strain NE82T were summed feature 8 (C18:1 ω7c and C18:1 ω6c) (71%) and C16:0 (7.9%), which also appeared in the R. frigidaeris JCM 32945T, P. ruber JCM 9931T and D. rubra JCM 30602T. C18:1 2-OH was another major fatty acid in R. frigidaeris JCM 32945T (10.0%). In addition, D. rubra JCM 30602T had another two types of fatty acid as main fatty acids, which were summed feature 3 (C16:1 ω7c and/or C16:1 ω6c) (18.5%) and C16:0 (14.0%). The detailed fatty acid compositions of strain NE82T and its closely related type strains are showed in Table 2.
The major polar lipids of strain NE82T were phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), an unidentified aminophospholipid (APL1) and an unidentified phospholipid (PL1) (Fig. S5). While the phosphatidylcholine (PC) was not detected in R. frigidaeris JCM 32945T, and there were four unidentified lipids (L1, L2, L3, L4) and six unidentified aminolipids (AL1, AL2, AL3, AL4, AL5, AL6) in R. frigidaeris JCM 32945T (Khan et al. 2019) . Besides, NE82T cells had also three another unidentified phospholipids (PL2, PL3 and PL4). The detailed comparisons were listed in Table 1. The sole menaquinone was Q-10, which was same with the closely related type strains.
According to all these results of phenotypic, biochemical and physiological analyses, together with the phylogenetic differences, strain NE82T can be assigned to the genus Roseicella within the family Acetobacteraceae, as representing a novel species, for which the name Roseicella aquatilis sp. nov. is proposed.
Description of Roseicella aquatilis sp. nov.
Roseicella aquatilis (a.qua’ti.lis. L. masc. adj. aquatilis living, growing or found in, or near, water, aquatic).
Cells are ellipsoidal, approximately 0.4-0.9 μm in diameter, Gram-stain-negative, non-motile and facultatively aerobic. Colonies are red-pigmented, circular and 1.0 mm in diameter after incubation at 30°C for four days. Cells are able to grow at 15-42°C, pH 5.5-8.5 and in the presence of 0-1.0% (w/v) NaCl and its optimal growth is at 28-33°C, pH 7.0-7.5, with 0% NaCl. Cells can reduce nitrate and are catalase positive, but oxidase negative. Tweens 20, 40, 60 are hydrolysed, but starch, casein, cellulose, alginate, and Tween 80 are not hydrolysed. Cells can produce alkaline phosphatase, esterase (C4), naphthol-AS-BI-phosphohydrolase, and leucine arylamidase, but the results for the production of esterase lipase (C8), lipase (C14), valine arylamidase, cycstine arylamidase, trypsin, ɑ-chymotrypsin, acid phosphatase, ɑ-galactosidase, β-galactosidase, β-glucuronidase, ɑ-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, ɑ-mannosidase, β-fucosidase are negative. Positive for citrate utilization, urease, but negative for o-nitrophenyl-β-D-galactopyranoside, ornithine decarboxylase, H2S production, indole production, Voges-Proskauer reaction and the tests of arabinose. Acids are produced from L-arabinose (weakly), D-Ribose (weakly), D-xylose (weakly), L-xylose (weakly), L-rhamnose (weakly), potassium gluconate (weakly)，potassium 5-ketogluconate (weakly), but not from glycerol, erythritol, D-arabinose, methyl-β-D-xylopyranoside, D-galactose, D-glucose, D-fructose, D-mannose and so forth. The sole menaquinone is Q-10. The main polar lipids are phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), an aminophospholipid (APL1), an unidentified phospholipids (PL1) and the dominant fatty acids are summed feature 8 (C18:1 ω7c and C18:1 ω6c).
The type strain, NE82T (＝KCTC 62412T＝MCCC 1H00292T), was isolated from Jiugongli Lake in Inner Mongolia Autonomous Region, China (106°49.721' E, 40°32.476' N). The DNA G+C content of the strain is 72.0 mol%. The GenBank accession numbers of strain NE82T for the 16S rRNA gene and genome sequences are
MG385132 and SKBM00000000, respectively.