Phylogenetic analysis and genome sequencing
Analysis of the 16S rRNA gene sequences revealed that strain G4T belonged to the genus Oceaniglobus, within the family Rhodobacteraceae. Strain G4T shared the highest sequence similarity with O. ichthyenteri (96.6%), followed by O.indicus (95.3%). The neighbour-joining phylogenetic tree (Fig. 1) revealed that strain G4T, O. ichthyenteri and O. indicus formed an monophyletic cluster, with high bootstrap support (99%). It indicated that strain G4T might represent a novel species of the genus Oceaniglobus.
The genome of strain G4T was comprised of 4472 genes and 33 contigs with a total length of 4588953 bp. The main coverage was 360 ×. The N50 value was 226458 bp. There were 5S rRNAs of 3, 16S rRNA of 2, 23S rRNAs of 2, tRNAs of 47 and sRNAs of 3. Complete genome analysis revealed that the 4,099 protein-coding genes constituted 91.7% of the total genes in the genome. Furthermore, there were 3,965 genes (88.7%) connected to KEGG pathways, 3,320 genes (74.2%) assigned to 24 different clusters of orthologous groups (COGs) and 1,458 genes (32.6%) connected to SwissProt pathways. Based on the genome sequence annotation, the genome contained several genes coding for Glycoside Hydrolases (GHs), Glycosyl transferases (GTs) and Carbohydrate esterases (CEs). There were 3965 genes annotated by KEGG pathway annotation, involving cellular Processes, environmental information processing, genetic information processing, human diseases, metabolism and organismal systems and a large number of genes were found to be focus on metabolism and membrane transport. The genes that encoded the enzymes required for PHB synthesis were present in the genome, the presence of genes encoding phaZ (EC126.96.36.199) and phaC (EC 2.3.1.-) showed the metabolic capacity of strain G4T to accumulate PHB intracellularly.
According to the genome sequence, the DNA G + C content was 65.3 mol%, which was higher than the related strains O. indicus (59.0 mol%) and O. ichthyenteri (64.2 mol%). The AAI values were 71.3% with O. indicus and 75.0% with O. ichthyenteri, which were far below the 90% cut-off value for prokaryotic species delineation (Rodriguez-R and Konstantinidis 2014). AAI values of 71.3% and 75.0% indicated that strain G4T represents a new species. The ANI values were 76.4% with O. indicus and 76.3% with O. ichthyenteri, which were lower than the 95–96% cut-off value for species demarcation (Richter and Rossello-Mora 2009). The dDDH values were 20.0% with O. indicus, 19.4% with O. ichthyenteri, which were below the standard cut-off value (70%) (Meier-Kolthoff et al. 2013). These results confirmed that strain G4T represented a novel species of the genus Oceaniglobus.
Morphology, physiology, and biochemical analysis
Cells were Gram-stain negative, aerobic, motile, gliding, lacking flagella, and oval (0.5–0.8 µm in width, 0.6–1.0 µm in length) (Fig. S1). Colonies were 0.8–1.0 mm in diameter, creamy, circular and convex with smooth surfaces after incubation on MA at 30°C for 2 days. NaCl was not essential for growth, which distinguished strain G4T from the related strains, O. ichthyenteri and O. indicus. The accumulation of PHB granules was observed by fluorescence microscopy. Besides, it was sensitive to ampicillin, penicillin, erythromycin, chloramphenicol, ciprofloxacin, gentamicin and cefazolin. Other phenotypic characteristics of the strain G4T and related strains are shown in Table 1.
The major fatty acid was summed feature 8 (C18:1ω7c and/or C18:1ω6c). The cellular fatty acid composition was similar to that of the related taxa of the genus Oceaniglobus. The detailed results of the fatty acids are shown in Table 2. The sole isoprenoid quinone found in strain G4T, ubiquinone-10 (Q-10), was in accordance with the properties of the genus Oceaniglobus. The polar lipid components of strain G4T were iphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidyldimethylethanolamine. Further detailed polar lipid images of the different strains are given in Fig. S1 (available in the online version of this article).