Morphological, physiological and biochemical characteristics
Cells of strain NBU1469T were Gram-negative, rod-shaped, non-sporulating and motile (Fig. 1). Colonies were 0.3 mm in diameter, circular, elevated and cream-colored after growing on MA at 26ºC for 3 days. Strain NBU1469T grew at 0.0-14.0% (w/v) NaCl (optimum 2.0%, w/v), pH 6.0-9.5 (optimum 7.5) and 20-40ºC (optimum 40ºC), but not at 45ºC. Strain NBU1469T was able to grow in MB containing high concentrations of heavy metals, including Cd2+ (0.5 mM), Cu2+ (5.0 mM), Mn2+ (2.0 mM), Zn2+(0.5 mM) or Co2+ (2.0 mM). No growth was detected under anaerobic conditions on modified MA with the addition of different electron acceptors even after two weeks. Other physiological and biochemical characteristics of strain NBU1469T are given in the species description.
The predominant fatty acid of strain NBU1469T were C16:0 (10.5%), C19:0 cyclo ω8c (27.0%) and summed feature 8 (50.5%). The complete fatty acid profiles are summarized in Table 2. Ubiquinone-10 (Q-10) was the only detected respiratory quinone. The polar lipids included phosphatidylglycerol (PG), two unidentified amino-phospholipids (PN), three unidentified phospholipids (PL) and two unidentified lipids (L) (Fig. S1).
Phylogenetic analysis and genome characterization
Based on the result of 16S rRNA gene sequence alignment, strain NBU1469T shared high sequence similarities of 98.1% and 97.6% to the species with validated name N. nitritireducens DR41_18T and N. denitrificans DR41_21T, respectively. Phylogenetic analysis revealed that strain NBU1469T was affiliated with N. nitritireducens DR41_18T and N. denitrificans DR41_21T on the different phylogenetic trees (Fig. 2, Fig. S2 and Fig. S3). Result of phylogenomic analysis also supported that strain NBU1469T closely related to N. nitritireducens DSM 19540T and N. denitrificans DSM 18348T (Fig. S4).
The draft genome sequence of strain NBU1469T and N. nitritireducens DSM 19540T were composed of 22 contigs for 5,022,713 bp with 63.6 mol% G+C content and 97 contigs for 4,845,838 bp with 60.6 mol% G+C content, respectively. The genome of strain NBU1469T contained 4,835 protein-coding genes and 77 RNA genes while N. nitritireducens DSM 19540T contained 4,506 protein-coding genes and 76 RNA genes. The result of antiSMASH 6.0 indicated that the novel isolate contained 1 gene cluster for terpenoid type, 1 gene cluster for ectoine type, 1 gene cluster for non-ribosomal peptide synthetase (NRPS) which is a multifunctional key enzyme in the process of biosynthesis of secondary metabolites which shown only 11% similarity to cupriachelin and 1 gene cluster belong to Type III PKS (polyketide synthase) (Fig. S5). The ANI and the dDDH values between strain NBU1469T and N. nitritireducens DSM 19540T were 78.8% and 21.1%, and the ANI and the dDDH values between strain NBU1469T and N. denitrificans DSM 18348T were 78.5% and 21.0%, which were well below the threshold value of 95% ANI relatedness for species delineation (Richter et al., 2009) and the proposed cut-off borderline of 70% (Wayne et al., 1987).
The experiments showed the strain NBU1469T could resist high concentrations of Cu2+ (5.0 mM) and Co2+ (2.0 mM). The adaption mechanism to heavy metal resistant was analyzed. According to the genome annotation results, strain NBU1469T harbored various putative proteins related to heavy metals resistance. Resistance to Cu2+ was mainly based on genes involved in copper homeostasis in the genome of strain NBU1469T. There were 13 genes involved in copper homeostasis: 3 multicopper oxidase, 1 cytochrome c heme lyase subunit CcmF, 2 copper-translocating P-type ATPase, 3 multidrug resistance transporter, Bcr/CflA family, 1 copper resistance protein B, 1 copper tolerance protein, 1 magnesium and cobalt efflux protein CorC and 1 copper homeostasis protein CutE (Fan et al., 2018). The genome of strain NBU1469T also contained 7 genes related to Co2+ uptake and resistance, containing 2 magnesium and cobalt transport protein CorA which help to uptake and accumulate Co2+, and 1 magnesium and cobalt efflux protein CorC which involved in the Co2+ efflux function (Wu et al., 2018). In addition, 3 MerR family regulators and 1 transcriptional regulator HmrR were found in the genome. These proteins were regulators of various environmental stimuli, particularly, under high concentrations of heavy metals and oxidative stresses (Wu et al., 2016). All above genes may contribute to the phenotype of strain NBU1469T in copper and cobalt resistance.
The major cellular fatty acids (C16:0, C19:0 cyclo ω8c and summed feature 8), predominant respiratory quinone (ubiquinone Q-10), major polar lipids (PG, PN1, PN2, PL1 and PL2), phylogenetic trees and phylogenomic tree supported that strain NBU1469T should be classified into the genus Nisaea. However, there are some additional differences between strain NBU1469T and the related type strains of genus Nisaea. The detailed fatty acid profile showed that strain NBU1469T possessed a higher amount of C19:0 cyclo ω8c than reference species, while possessing lower amount of summed feature 3 and 8 than reference species (Table 2). The detailed polar lipid profile showed strain NBU1469T contained PL3, while not in strain N. nitritireducens DSM 19540T (Fig. S1). L1, L2, L3 and L4 were detected in strain N. nitritireducens DSM 19540T but not in strain NBU1469T and N. denitrificans DSM 18348T. Among these three strains, only NBU1469T possessed L5 and L6. The DNA G+C content of strain NBU1469T (63.6 mol%) was higher than that of N. nitritireducens DSM 19540T (60.6 mol%) and N. denitrificans DSM 18348T (60.0 mol%). There were also several phenotypic differences between NBU1469T and related type strains. Strain NBU1469T could tolerate NaCl concentration up to 14.0%, but two type strains could not grow at NaCl concentration higher than 6.0%. Hydrolysis of tween 20 and nitrate reduction were positive for NBU1469T but negative for two type strains (Table 1). Among these three species, only strain N. denitrificans DSM 18348T was facultatively anaerobic. Meanwhile, the ANI values (78.5-78.8%) and dDDH values (21.0-21.1%) between strain NBU1469T and the two reference species were below the thresholds recommended for species delineation 95% (ANI) and 70% (dDDH). All of the above confirmed that strain NBU1469T represented a novel species within the genus Nisaea.
Based on the phenotypic, chemotaxonomic and genotypic characteristics described above, we identified strain NBU1469T as the type strain of a novel species of the genus Nisaea, for which the name Nisaea sediminum sp. nov. is proposed.
Description of Nisaea sediminum sp. nov.
Nisaea sediminum (se.di.mi’num. L. gen. pl. n. sediminum of sediments, pertaining to source of the isolate).
Cells are Gram-negative, aerobic, rod-shaped and motile. The cell size is 0.4-1.0×0.9-2.9 μm. Colonies on Marine agar 2216 are 0.3 mm in diameter, circular, elevated and cream-colored after 3 days 26ºC. The temperature range for growth is 20-40ºC (optimum 40ºC), but not at 45ºC. Growth occurs at 0-14.0% NaCl and pH 6.0-9.5 (optimum, 2% NaCl and pH 7.5). Positive for catalase and oxidase activities, nitrate reduction, H2S production and hydrolysis of Tween 20. Negative for methyl red, indole production, urease, fermentation of D-glucose, arginine dihydrolase, β-galactosidase, hydrolysis of starch, casein, gelatin, aesculin, Tweens 40, 60 and 80. In the API ZYM kit, positive for activity of alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valin arylamidase, acid phosphohydrolase and naphtol-AS-BI-phosphohydrolase. In the API 50CH kit, positive for acid production from glycerol, D-arabinose, L-xylose, D-galactose, L-fucose, D-Ribose, D-lyxose, L-Arabinose, D-xylose, L-rhamnose, D-fucose, 2-ketogluconate and 5-ketogluconate. Sensitive to gentamicin, cefradine, clindamycin, nalidixic acid, vancomycin, oxacillin, doxycycline, neomycin, kanamycin, ofloxacin, amikacin, rifampicin, novobiocin and minocycline. The major fatty acid are C16:0, C19:0 cyclo ω8c and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). Q-10 is the only detected respiratory quinone. The polar lipids include phosphatidylglycerol, two unidentified amino-phospholipids, three unidentified phospholipids and two unidentified lipids. The DNA G+C content of the genomic DNA of the type strain is 63.6 mol%.
The type strain NBU1469T (=KCTC 82224T=MCCC 1K04763T) was isolated from a marine sediment sample taken from the Meishan Island in the East China Sea. The GenBank accession numbers for the 16S rRNA gene and the draft genome data of strain NBU1469T are MT525301 and JACZCQ000000000, respectively.