Nisaea sediminum sp. nov., a heavy metal resistant bacterium isolated from marine sediment in the East China Sea

A Gram-negative, rod-shaped, motile and strictly aerobic bacterium, designated NBU1469T, was isolated from marine sediment sampled on Meishan Island located in the East China Sea. Strain NBU1469T grew optimally at temperature of 40 °C, NaCl concentration of 2.0% (w/v) and pH 7.5. Catalase and oxidase activities, H2S production, nitrate reduction and hydrolysis of Tween 20 were positive. Indole, methyl red reaction, urease, hydrolysis of gelatin, starch, casein, Tweens 40, 60 and 80 were negative. The major cellular fatty acids were C16:0, C19:0 cyclo ω8c and summed feature 8 (C18:1ω7c and/or C18:1ω6c). The only respiratory quinone was ubiquinone-10 (Q-10). The major polar lipids were phosphatidylglycerol, two unidentified amino-phospholipids and two unidentified phospholipids. Comparative analysis of the 16S rRNA gene sequence showed highest similarities to the species with validated name Nisaea nitritireducens DR41_18T (98.1%) and Nisaea denitrificans DR41_21T (97.6%). Phylogenetic analyses indicated that strain NBU1469T formed a distinct lineage with strains Nisaea nitritireducens DR41_18T and Nisaea denitrificans DR41_21T within the genus Nisaea. The average nucleotide identity and digital DNA-DNA hybridization values between strain NBU1469T and related species of genus Nisaea were well below the threshold limit for prokaryotic species delineation. The DNA G + C content was 63.6%. Based on its phenotypic, chemotaxonomic and genotypic data, strain NBU1469T is considered to be a representative of a novel species in the genus Nisaea, for which the name Nisaea sediminum sp. nov. is proposed. The type strain is NBU1469T (=KCTC 82224 T =MCCC 1K04763T).


Introduction
The genus Nisaea, belonging to the family Rhodospirillacea in the class Alphaproteobacteria, was originally proposed by Urios et al. (2008) with the description of Nisaea nitritireducens DR41_18 T and Nisaea denitrificans DR41_21 T . At the time of writing, the genus Nisaea only contained the above two validly published species (https://www.bacterio. net/genus/Nisaea), which were reported from coastal, surface waters (Urios et al. 2008). Cells of N. nitritireducens DR41_18 T and N. denitrificans DR41_21 T were reported as Gram-negative, motile, rod-shaped, catalase-and oxidase-positive, containing ubiquinone-10 (Q-10) as the predominant quinone, with very high amounts of C 18:1 x7c and the DNA G ? C content was around 60%. Genus Nisaea, identified as hubs in most of subnetworks of microbial communities, may have the potential to synchronize ecological processes over broad ecosystems (Ma et al. 2020). In this paper, we describe a novel strain, designated NBU1469 T , isolated from a marine sediment sample collected from Meishan Island in the East China Sea. Following the polyphasic taxonomic approach, we propose that strain NBU1469 T represents a novel species of the genus Nisaea.

Material and methods
Bacterial strains and culture condition Sediment samples were collected from the Meishan Island located in the East China. Sea, Ningbo, China (121°56 0 E, 29°45 0 N) in August 2019. Marine broth 2216 (MB, Difco) was used for isolation. The medium was solidified with 2.0% agar (MA). About 3.0 g sediment sample was serially diluted to 10 -4 with MB, and 200 ll of each diluted sample was spread on MA plates. After 3 days of incubation at 26°C, a creamcolored colony was picked and purified by restreaking. The isolate was routinely cultured in MB at 26°C and stored at -80°C with 25% (v/v) glycerol. The type strains used for reference Nisaea nitritireducens DSM 19540 T and Nisaea denitrificans DSM 18348 T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ; Germany). Two type strains were cultured under the identical experimental conditions as strain NBU1469 T for comparative analysis.
Morphological, physiological and biochemical characterization Cell morphology was observed by using an optical microscope (BX40; Olympus) and transmission electron microscopy (JEM-1230; JEOL). Exponentially growing cells incubated on MA plates were suspended and stained with uranyl acetate and then fixed on the copper mesh before observed with transmission electron microscopy. Gram staining was performed according to Dong and Cai (2001). Motility was examined by microscopic observation and inoculation on semi-solid MB medium with 0.5% agar (w/v). To determine the growth conditions of strain NBU1469 T , the temperature range for growth was determined in MB at 4,10,15,20,25,28,30,35,37,40,41,42,43,44,45,50 and 55°C. The pH range for growth was determined at pH 4.0-10.0 (at intervals of 0.5) in MB supplemented with the following buffers: ammonium acetate (pH 4.0-5.0), MES (pH 5.5-6.0), PIPES (pH 6.5-7.0), Tricine (pH 7.5-8.5) and CAPSO (pH 9.0-10.0) at a concentration of 30 mM. The NaCl concentration range for growth was determined at 0, 0.5 and 1-10.0% (at intervals of 1%, w/v) NaCl in modified MB (with original Na ? and Clremoved). All tests of growth conditions were performed in quadruplicate and OD 600 measurements were taken after 24 h incubation at 26°C with shaking at 140 rpm.

Chemotaxonomic characterization
Biomass for chemotaxonomic and molecular studies was obtained by cultivation in MB at 26°C for 3 days, with shaking at 140 rpm. All the following tests for chemotaxonomic characterization were performed on strains NBU1469 T , N. nitritireducens DSM 19540 T and N. denitrificans DSM 18348 T under the same conditions. The identification and quantification of fatty acid methyl esters (FAMEs) were performed using the Sherlock Microbial Identification System (MIDI) with the standard MIS Library Generation Software version 6.1 according to the manufacturer's instructions. Respiratory quinones were extracted and analyzed by using reversed-phase HPLC as described by Minnikin et al. (1984). Total lipids were extracted as described by Kates (1986) and detected by twodimensional TLC silica-gel 60 F 254 aluminiumbacked thin-layer plates (10 9 10 cm, Merck 5554), and further analyzed as described by Minnikin et al. (1984). The TLC plates were sprayed with phosphomolybdic acid with 5% in ethanol to reveal total lipids, a-naphthol/H 2 SO 4 to reveal glycolipids, molybdenum blue to reveal phospholipids and ninhydrin to reveal aminolipids (Zhang et al. 2015).
The whole genomes of strains NBU1469 T and Nisaea nitritireducens DSM 19540 T were sequenced using an Illumina HiSeq 4000 system (Illumina) at the Beijing Genomics Institute (Shenzhen, China) (Zhang et al. 2020). The paired-end fragment libraries were sequenced according to the Illumina HiSeq 4000 system's protocol. Raw reads of low quality from paired-end sequencing (those with consecutive bases covered by fewer than five reads) were discarded. The sequenced reads were assembled using SOAPdenovo v1.05 software (Li et al. 2008). The protein coding sequences (CDSs) were annotated by using Rapid Annotation using Subsystem Technology (RAST) server online (Overbeek et al. 2014). The antiSMASH 6.0 program was used as a tool for the identification of the secondary metabolism gene clusters (Kai et al. 2019). Genome data publicly available of Nisaea denitrificans DSM 18348 T (AUFM00000000) was retrieved from the NCBI Genome database. The average nucleotide identity (ANI) values between strain NBU1469 T and two reference species were calculated using the ANI calculator online service (Yoon et al. 2017a, b). Digital DNA-DNA hybridization (dDDH) values were calculated by the genometo-genome distance calculator (GGDC) server version 2.1 (Meier-Kolthoff et al. 2013).

Results and conclusion
Morphological, physiological and biochemical characteristics Cells of strain NBU1469 T were Gram-negative, rodshaped, non-sporulating and motile (Fig. 1). Colonies were 0.3 mm in diameter, circular, elevated and cream-colored after growing on MA at 26°C for 3 days. Strain NBU1469 T grew at 0-14.0% (w/v) NaCl (optimum 2.0%, w/v), pH 6.0-9.5 (optimum 7.5) and 20-44°C (optimum 40°C), but not at 45°C. Strain NBU1469 T was able to grow in MB containing high concentrations of heavy metals, including Cd 2? (0.5 mM), Cu 2? (5.0 mM), Mn 2? (2.0 mM), Zn 2? (0.5 mM) or Co 2? (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 NBU1469 T are given in the species description (Table 1).

Phylogenetic analysis and genome characterization
Based on the result of 16S rRNA gene sequence alignment, strain NBU1469 T shared high sequence similarities of 98.1% and 97.6% to the species with validated name N. nitritireducens DR41_18 T and N. denitrificans DR41_21 T , respectively. Phylogenetic Fig. 1 Electron micrographs of cells of strain NBU1469 T growing on MA medium, cells are rod-shaped with a monopolar flagellum. Bar, 500 nm analysis revealed that strain NBU1469 T was affiliated with N. nitritireducens DR41_18 T and N. denitrificans DR41_21 T on the different phylogenetic trees (Fig. 2,  Fig. S2 and Fig. S3). Result of phylogenomic analysis also supported that strain NBU1469 T closely related to N. nitritireducens DSM 19540 T and N. denitrificans DSM 18348 T (Fig. S4).

API 50CH
Glycerol, D -arabinose, L -xylose, D -galactose, L -fucose, 5-ketogluconate  Strains: 1, strain NBU1469 T ; 2, N. nitritireducens DSM 19540 T ; 3, N. denitrificans DSM 18348 T . All data were taken from this study unless otherwise indicated. Data marked with a was taken from Urios et al. (2008). -, negative; ? , positive; R, resistant; S, susceptible. The same characteristics shared by these three strains were listed in Table S1 strains NBU1469 T and N. nitritireducens DSM 19540 T were 78.8% and 21.1%, and the ANI and the dDDH values between strains NBU1469 T and N. denitrificans DSM 18348 T were 78.5% and 21.0%, respectively, 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) (Table S2). The experiments showed the strain NBU1469 T as well as two reference strains could resist high concentrations of Cu 2? (5.0 mM) and Co 2? (2.0 mM). The adaption mechanism to heavy metal resistant was analyzed. According to the genome annotation results, strain NBU1469 T harbored various putative proteins related to heavy metals resistance. Resistance to Cu 2? was mainly based on genes involved in copper homeostasis in the genome of strain NBU1469 T . There were 13 genes involved in copper homeostasis: 3 multicopper oxidase, 1 cytochrome c heme lyase subunit CcmF, 2 coppertranslocating 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 NBU1469 T also contained 7 genes related to Co 2? uptake and resistance, containing 2 magnesium and cobalt transport protein CorA which help to uptake and accumulate Co 2? , and 1 magnesium and cobalt efflux protein CorC which involved in the Co 2? 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 NBU1469 T in copper and cobalt resistance.
Compared to strain NBU1469 T , the genome annotation results showed two reference strains contained similar putative proteins related to heavy metals resistance, including multicopper oxidase, cytochrome c heme lyase subunit CcmF, copper-translocating P-type ATPase, multidrug resistance transporter, Bcr/CflA family, copper resistance protein, magnesium and cobalt efflux protein CorC and copper homeostasis protein CutE which related to copper homeostasis;magnesium and cobalt transport protein CorA, magnesium and cobalt efflux protein CorC, MerR family regulators and transcriptional regulator HmrR which referred to Co 2? uptake and resistance.

Taxonomic conclusion
The major cellular fatty acids (C 16:0 , C 19:0 cyclo x8c 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 NBU1469 T should be classified into the genus Nisaea. However, there are some additional differences between strain NBU1469 T and the related type strains of genus Nisaea. The detailed fatty acid profile showed that strain NBU1469 T possessed lower amount of summed feature 3 than reference species (Table 2). The detailed polar lipid profile showed strain NBU1469 T contained  (Fig. S1). L1, L2, L3 and L4 were detected in strain N. nitritireducens DSM 19540 T but not in strain NBU1469 T and N. denitrificans DSM 18348 T . The DNA G ? C content of strain NBU1469 T (63.6%) was higher than that of N. nitritireducens DSM 19540 T (60.6%) and N. denitrificans DSM 18348 T (60.5%). There were also several phenotypic differences between NBU1469 T and related type strains. Strain NBU1469 T 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 was positive for NBU1469 T but negative for two type strains (Table 1). Among these three species, only strain N. denitrificans DSM 18348 T was facultatively anaerobic. Meanwhile, the ANI values (78.5-78.8%) and dDDH values (21.0-21.1%) between strain NBU1469 T 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 NBU1469 T represented a novel species within the genus Nisaea. Based on the phenotypic, chemotaxonomic and genotypic characteristics described above, we identified strain NBU1469 T as the type strain of a novel species of the genus Nisaea, for which the name Nisaea sediminum sp. nov. is proposed.
The type strain NBU1469 T (=KCTC 82224 T-=MCCC 1K04763 T ) 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 NBU1469 T are MT525301 and JACZCQ000000000, respectively.