Genomic features and analysis
The assembled genome of strain HB171785T was 4320414 bp in size, with 46 contigs and N50 177479 bp. The sequence depth of coverage was 145× and DNA G + C content was 49.7%. A total of 3746 genes including 3668 coding genes, 6 rRNAs (3, 1, 2 for 5S, 16S and 23S rRNA, respectively) and 56 tRNAs were detected (Table S1). Following the annotated genome of strain HB171785T in KEGG, it includes environmental information processing, genetic information processing, cellular processes, and metabolism. Most of the proteins were mainly affiliated with metabolism of biosynthesis of amino acids, carbon metabolism, and ABC transporters. In clusters of orthologous groups (COGs) functional classes, the most abundantly represented coding sequences of strain HB171785T were mainly involved in amino acid transport and metabolism, cell wall/membrane/envelope biogenesis, carbohydrate transport and metabolism, inorganic ion transport and metabolism, and cell motility.
The bacterial core genome tree was generated with 92 single-copy and homologous genes using UBCG tool. The phylogenomic tree (Fig. 2) showed that strain HB171785T formed a clade with N. marina j221T, and close to “N. holothuriorum” 126 and E. agarilytica LMG2520T, which was consistent with Fig. 1. Calculation of the gene support index indicated that 81 genes supported the branch between strain HB171785T and N. marina j221T in the concatenated phylogenetic tree. The novel isolate exhibited ANI values of 71.4–82.9%, and dDDH values (the recommended results from formula 2) of 19.8–25.4% with the three closest relatives (Table S2), which were strikingly lower than the threshold values of the species boundary (ANI 94–96%, and dDDH 70%) (Chun et al. 2018). The low taxonomic relatedness and the phylogenomic tree suggested that strain HB171785T represents a different genomic species of genus Neiella.
To search for genes related to polysaccharide-degrading enzymes in the genomes, the carbohydrate-related genes were annotated on the basis of the CAZyme database. CAZymes are 216, 180, 126, 181 and the proportions are about 5.9, 5.0, 3.6 and 5.2% in HB171785T, N. marina j221T, N. holothuriorum 126T and E. agarilytica KMM 6351T, respectively. Generally speaking, CAZymes seldom exceed 5% of the bacterial genomes that engage in carbohydrate degradation, while they rarely exceed 2% in most bacterial genomes (Mann et al. 2013). Further analysis revealed that the putative polysaccharide lyases (PLs) were involved in alginate degradation in all the four bacteria. With the exception of HB171785T, the three bacteria of order Alteromonadales were isolated from intestine of sea cucumber and sea urchin which feed on alga, and might produce abundant polysaccharide lyases.
Taxonomic conclusions
The results of physiological features (cell size; the optimum temperature for culture, flagellum shape, activities of α-galactosidase, β-galactosidase and catalase, et al.), fatty acid profiles, ANI and dDDH values clearly differentiate strain HB171785T from the closest neighbors. Therefore, on the basis of the phenotypic, chemotaxonomic, phylogenetic and genetic data, strain HB171785T is considered to represent a novel species of the genus Neiella, for which the name Neiella litorisoli sp. nov. is proposed.
Description of Neiella litorisoli sp. nov.
Neiella litorisoli sp. nov. (li.to.ri.so'li. L. neut. n. litus, -oris, seashore; L. neut. n. solum, soil; N.L. gen. n. litorisoli from soil of a seashore, referring to the source of isolation).
Cells are Gram-stain-negative, aerobic, non-spore-forming and motile by means of one to several polar or lateral flagella. Cells are rod-shaped with 2.2–4.5 µm in length and 0.4–0.7 µm in width. Colonies of strain HB171785T are circular, low convex, 2–3 mm in diameter, with regular edges, creamy white in colour after grown on MA after 48 h at 28°C. The temperature range for growth is 15–50°C (optimum, 37–40°C). The pH range for growth is 6–10 (optimum, pH 7–8). Growth occurs at 0.5–8% (w/v) NaCl (optimum, 3–4%). Positive for Voges–Proskauer test, catalase and alginate lyase activities; but negative for ethyl red test and H2S production. Indole was not produced and nitrate was not reduced to nitrite.
In the API 20NE test, hydrolysis of aesculin, β-galactosidase and oxidase are positive, and all the other characteristics are negative. In the API ZYM kit, tests for esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI- phosphohydrolase, α-galactosidase, β-galactosidase and β-glucosidase are positive, but negative for alkaline phosphatase, lipase (C14), trypsin, a-chymotrypsin, β-glucuronidase, a-glucosidase, N-acetyl-glucosaminidase, α-mannosidase and α-fucosidase. With API 50CHE strips, acid is not produced from all the carbon sources. Susceptible to penicillin, ampicillin, cephalexin, cefradine, cefuroxime, ceftazidime, ceftriaxone, cefoperazone, amikacin, gentamicin, kanamycin, neomycin, erythromycin, mideamycin, vancomycin, co-trimoxazole, chloramphenicol and clindamycin; but resistant to cefazolin, doxycycline, tetracycline, minocycline, ofloxacin, ciprofloxacin, polymyxin and distillers. The predominant respiratory quinone is Q-8. The major polar lipids are diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Major cellular fatty acids (> 5%) are C16:0, C16:1 ω7c and/or C16:1 ω6c, C18:1 ω7c and/or C18:1 ω6c, and C12:0 3OH.
The type strain is HB171785T (= MCCC 1K04625T = KCTC 82319T), isolated from a soil sample collected from Qishui Bay, Hainan, PR China. The DNA G + C content of the type strain is 49.7%. The GenBank/EMBL/DDBJ accession numbers for 16S rRNA gene and complete genome sequences are MN911325 and JACXAF000000000, respectively.