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). The genome of E. agarilytica LMG2520T was sequenced with 26 contigs and 149× coverage depth. Genomic analyses showed that strain LMG2520T presented a genome of 4236138 bp with chromosomal G + C content of 46.0%. A total of 3623 genes, including 3507 protein-coding, 6 rRNAs and 61 tRNAs were predicted.
In the phylogenomic tree (Fig. S3), strain HB171785T clustered with Neiella marina j221T, “Neiella holothuriorum” 126T and Colwellia chukchiensis CGMCC 1.9127T (Yu et al. 2011), close to Echinimonas agarilytica LMG2520T, whereas it clustered in a subclade with the three strains in the 16S phylogenetic tree except for C. chukchiensis. The novel isolate exhibited ANI values of 75.2–82.9%, dDDH values (the recommended results from formula 2) of 19.8–25.4% and AAI values of 77.1–89.2% with the closest relatives of N. marina and “N. holothuriorum”, respectively (Table S2). All of these values are strikingly lower than the threshold values of the species boundary (ANI 94–96%, dDDH 70% and AAI 95%) (Chun et al. 2018), indicating that the isolate does not belong to any of these species of genus Neiella. Meanwhile, strain HB171785T exhibited 16S rRNA gene similaritis of 89.8–95.0%, ANI values of 66.7–71.4%, dDDH values of 19.5–20.9% and AAI values of 51.3–68.5% with E. agarilytica and C. chukchiensis, respectively. An AAI ≤ 74% indicates that two strains represent members of different genera (Nicholson et al. 2020), which means that the novel strain does not belong to genera of Echinimonas and Colwellia.
A total of 276 proteins were matched with the CAZy database, including 131 glycoside hydrolases, 66 glycosyl transferases, 35 polysaccharide lyases, 18 carbohydrate esterases, 11 carbohydrate-binding modules and 4 auxiliary activities. Further analysis revealed that ten of the putative polysaccharide lyases (PLs) were involved in alginate degradation and were classified into five families: 3 PL6, 3 PL7, 1 PL14, 2 PL17 and 1 PL31.
Chemotaxonomic characterization
Strain HB171785T contained C16:0 (28.7%), C16:1 ω7c and/or C16:1 ω6c (27.5%), C18:1 ω7c and/or C18:1 ω6c (24.9%) as the predominant cellular fatty acids (> 20%) (Table S3), which also predominated in the other species of genus Neiella and E. agarilytica KMM 6351T. The novel strain had relatively lower C18:0 and higher C12:0 3OH than the other closely relatives; and lacked C18:1 ω9c while the other two species of genus Neiella had moderate content. Meanwhile, major cellular fatty acids (> 4%) of Colwellia chukchiensis CGMCC 1.9127T were iso-C15:0 2-OH and/or C16:1 ω7c 2-OH (28.1%), C16:0 (13.3%), C17:1 (12.9%), C15:1 (8.6%), iso-C16:0 (6.7%), C15:0 (5.8%), C16:1 ω9c (4.8%) and C17:0 (4.2%) (Yu et al. 2011), which were quite different from strain HB171785T. The major respiratory quinone was identified as Q-8. The major polar lipids included diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) (Supplementary Fig. S4). The characteristics are basically consistent with those of the genus Neiella (Du et al. 2013).
Taxonomic conclusions
The results of physiological features (cell size; the optimum temperature for culture, activities of alginate lyase, α-galactosidase, β-galactosidase and catalase, et al.), fatty acid profiles, ANI, dDDH and AAI 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 Paenibacillus arenilitoris sp. nov.
Neiella litorisoli sp. nov. (li.to.ri.so'li. L. n. litus, -oris, seashore; L.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. 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.