Halomonas Salinarum sp. nov., a Moderately Halophilic Bacterium, Isolated from Saline Soil in Yingkou, China

doi:10.21203/rs.3.rs-1005992/v1

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Halomonas Salinarum sp. nov., a Moderately Halophilic Bacterium, Isolated from Saline Soil in Yingkou, China

https://doi.org/10.21203/rs.3.rs-1005992/v1

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Strains of Halomonas, thought to play vital roles in the environment for their versatility, are ubiquitous in hypersaline environments. A Gram-staining-negative, moderately halotolerant, facultatively aerobic, motile bacterium, designated G5-11^T, was isolated from saline soil in Yingkou of Liaoning, China. The cells of strain G5-11^T grew at 4-35 ℃ (optimum 30 ℃), at pH 6.0-9.0 (optimum 8.0) and in the presense of 3-15 % (w/v) NaCl (optimum 5 %). The strain could be clearly distinguished from the related species of the genus Halomonas by its phylogenetic position and biochemical characteristics. It presented Q-9 as the major respiratory quinone and the dominant cellular fatty acids were summed feature 8 (C_18:1 ω7c/ C_18:1 ω6c), C_16:0 and summed feature 3 (C_16:1 ω7c/ C_16:1 ω6c). The polar lipids consisted of phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol as the major components. The G+C content of strain G5-11^T genome was 61.0 mol%. 16S rRNA analysis showed that strain G5-11^T had the highest similarity to Halomonas niordiana LMG 31227^T and Halomonas taeanensis DSM 16463^T, both reaching 98.3 %, followed by Halomonas pacifica NBRC 102220^T with a value of 95.8 %. Based on phenotypic, chemotaxonomic and phylogenetic inferences, strain G5-11^T represents a novel species of the genus Halomonas, for which the name Halomonas salinarum sp. nov. is proposed. The type strain of Halomonas salinarum is G5-11^T (=CGMCC 1.12051^T=LMG 31677^T).

General Biochemistry

Molecular Biology

Internal Medicine

Halomonas salinarum sp. nov. Marine bacteria Moderately halotolerant alkaliphilic

The genus Halomonas was placed into the family Halomonadaceae of the class Gammaproteobacteria, as proposed by Vreeland et al. (1980a) and then emended by Dobson and Franzmann (1996). At the time of writing, the genus comprised 113 species with validly published names (http://www.bacterio.net/halomonas.html). The Halomonas was initially isolated from the saturation ponds and crystallizes in the facility and was in close association with extremely halophilic archaebacteria. Their extreme salt tolerance, the fact that they can survive, and prosper with very little NaCl suggested that they were present in virtually any saline environment, although recently they had also been found associated with plant rhizosphere (Kämpfer et al. 2018) and inhabiting plant stems (Chen et al. 2018a). In terms of their ability to successfully grow in a variety of temperatures and pH conditions, this versatility may eventually lead to Halomonas species using as a substitute for starch-derived raw materials utilization (Quillaguamán et al. 2004). Some species of the genus Halomonas had been recognized for potential applications in biotechnology due to their ability to produce exopolysaccharide (Amjres et al. 2011), produce compatible solutes and extracellular enzymes (Sanchez-Porro et al. 2003), degrade aromatic compounds (García et al. 2004) and finish biological nitrogen removal (Qu et al. 2011).

Yingkou saltworks, locates in the Northeastern China, is a essential industrial base in the Liaoning province which has abundant halophilic or halt-tolerant microbes. The unique environment of high salinity and high osmosis determines the cellular functional physiology and metabolic mechanism of these organisms as well as the particularity of community structure (Oren 2002, 2008). Due to their active substances with special structures and functions accumulated for tolerating hypersaline environments, halophilies are not only an important part of food chain in high-salt ecosystem, but also have broad application prospects in many fields. (etc. aquaculture, environmental governance, and biotechnology). In the course of investigating the microbial diversity of saline soil in Liaoning, a moderately halotolerant strain designated G5-11^T was found, which can grow under wide range of salt concentration and confirmed that the strain represents a novel species of the genus Halomonas based on phenotypic, molecular and chemotaxonomic evidence.

Isolation of bacterial strain and culture conditions

The strain was isolated from Yingkou, Liaoning, PR China (40°28′N 122°12′E) in July 2010. The pH of the saline soil was 8.0-8.5, with the 0.6-5.0 % salinity. For the isolation, a sample of saline soil was suspended in aseptic salines [containing 5 % (w/v) NaCl], and plated onto Gibbson medium containing (L⁻¹): 10 g yeast extract powder, 5.0 g casein, 5.0 g peptone, 3.0 g trisodium citrate, 20.0 g MgSO₄·7H₂O, 2.0 g KCl, 50 g NaCl and 15 g agar (Xu et al. 1995). The pH of the basal medium ager was 8.0. After incubation at 30 ℃ for 5 days, several single colonies were transferred onto new Gibbson agar plates; this step was repeated several times until a single colony purified by subculture. The pure culture was maintained at -80 ℃ in 30 % glycerol. Unless otherwise indicated, morphological, physiological and biochemical characteristic studies were performed with cells grown on Gibbson medium supplied with 5 % (w/v) NaCl at pH 8.0 and 30 ℃. Since the similarity of 16S sequence between G5-11^T and H. niordiana LMG 31227^T was less than 98.7 % (Chun et al. 2018), the data were quoted in the same culture conditions with the same methods. The type strain Halomonas taeanensis DSM 16463^T (= KCTC 12284^T) was obtained from the German Culture Collection (DSMZ, Braunschweig, Germany) was used for comparative purposes.

Phylogenetic and genotypic analysis

Amplification of the target strain 16S rRNA gene was performed using universal primer pair 27F/1492R (5′-AGAGTTTGATCCTGGCTCAG-3′/5′-TACGGYTACCTTGTTACGACTT-3′) and cloned into pMD-18 T vector (TaKaRa) for sequencing (Cui et al. 2001). The 23S rRNA sequence used for the phylogenetic tree and network database was obtained by de novo prediction of the whole genome. The phylogenetic relationship was determined after comparing the 16S rRNA gene sequences in EzBioCloud server databases (Yoon et al. 2017). Alignment the sequence of strain G5-11^T was carried out by using the software CLUSTAL_X version 2.0.10 with members of the genus Halomonas and other related taxa (Thompson et al. 1997). Phylogenetic analysis of the retrieved sequences was performed using the neighbor-joining (Saitou and Nei 1987), minimum-evolution (Rzhetsky and Nei 1993) and maximum-likelihood (Felsenstein 1992) algorithms by using the MEGA X (Kumar et al. 2018). Evolutionary distance matrices of phylogenetic trees were calculated according to Kimura’s two-parameter model (Kimura 1980). The topology of each tree was evaluated by bootstrap analysis of 1000 replications (Felsenstein 1985).

Bacterial Genomic DNA Rapid Extraction Kit (Beijing Huitian Oriental Technology Co., Ltd.) was used to extract the genome of strain G5-11^T according to the manufacturer’s instructions (Liu et al. 2016). The whole genome of strain G5-11^T was sequenced using Illumina NovaSeq PE150 apparatus at Beijing Novogene Bioinformatics Technology Co., Ltd.. SOAP denovo version 2.04 and Gap Closer version 1.12 were used for genome assembly and gap filling (Li et al. 2010). Gene prediction performed using GeneMarkS 4.17 (Besemer et al. 2001). The general functional databases GO, KEGG, NR, Pfam and Swiss- Prot were used to analyse the functional genes. The tRNAscan-SE (version 1.3.1) (Lowe and Eddy 1997) and RNAmmer (version 1.2) (Lagesen et al. 2007) were used to identificate tRNAs and rRNAs. To further confirm the phylogenetic status of G5-11^T, phylogenetic relationships of the genomes were explored with UBCG (Na et al. 2018) using default settings. The whole genome sequences of the reference strains in the phylogenomic tree were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/genome/). The average nucleotide identity (ANI) values were calculated by EzBioCloud online tool (https://www.ezbiocloud.net/tools/ani) (Lee et al. 2016) while the digital DNA–DNA hybridization (dDDH) values were calculated by using the Genome-to-Genome Distance Calculator available at https://ggdc.dsmz.de/ (Meier-Kolthoff et al. 2013). Formula 2 was applied for the dDDH analysis.

Based on its slightly halophilic and alkaliphilic nature, futher analysis of the gene/gene clusters of G5-11^T was performed. Compared the amino acid sequence of the target species with the TCDB (Saier et al. 2016), NR (Non-Redundant Protein Database), Swiss-Prot and KEGG database (Kanehisa et al. 2016) by Diamond software (Benjamin et al. 2015), combined the strain gene with its corresponding functional annotation information to obtain the annotation result. To ensure its biological significance, retained an optimal comparison result as the annotation of the gene.

Phenotypic and biochemical characterisation

Cell morphology of strain G5-11^T after 2 days of growth was examined by using transmission electron microscopy (JEM01230, JEOL). Samples for transmission electron microscopy were prepared as described by Ming et al. (2012). Gram-staining performed as described by Smibert and Krieg (1994). Motility of cells was examined on semisolid agar (Leifson 1960). Facultative anaerobic activity was determined by incubating in Gibbson liquid medium for 7 days and observing the distribution of strain G5-11^T. A range of phenotype characteristics of strain G5-11^T were tested together with the type strain H. taeanensis DSM 16463^T. Growth at various temperatures (0, 4, 10, 15, 20, 30, 35, 37 and 45 ℃) was investigated for 7 days. The pH range for growth was tested from pH 5.0 to 11.0 (at 1.0 pH unit intervals) using the buffers described by Xu et al (Xu et al. 2005). To test NaCl tolerance, growth at various concentrations of NaCl (1, 3, 5, 7, 10, 15 and 25 %, w/v). Catalase activity and oxidase activity were tested by assessing the formation of bubbles on the addition of 3 % (v/v) H₂O₂ and oxidation of tetramethyl-p-phenylenediamine (Kovacs 1956). Antibiotic susceptibility tested as described by Chen (Chen et al. 2018b) after incubation at 30℃ at 5 % salt concentration for 2 days on MA medium. Physiological and biochemical characteristics were examined using GEN III MicroPlates (Biolog), API 50 CHB, API ZYM and API 20NE strips (bioMérieux) according to the manufacturers’ instructions. All suspension media were supplemented with 5 % (w/v) NaCl and incubated at 30 ℃. To further confirm the results, the experiments were carried out in triplicate.

Chemotaxonomic characterisation

The respiratory quinones were extracted from freeze-dried cells (Collins et al. 1977) and analysed by HPLC (Tamaoka 1986). Polar lipids profiles of strain G5-11^T were extracted, separated and analysed by a two-dimensional TLC according to Minnikin et al. (1984). Cellular fatty acids were methylated, separated and identified with the Sherlock Microbial Identification System (MIDI, Sherlock version 6.0B) (Sasser 1990).

Phenotypic characteristics

Cells of strain G5-11^T were Gram-staining-negative, facultatively aerobic, motile utilizing peritrichous flagella and rod-shaped. (Fig. 1). On the surface of Gibbson medium with 5% NaCl, colonies were creamy, smooth and slightly irregular after incubation for 2 days at 30 ℃. Positive for D-maltose, D-trehalose, sucrose, D-turanose, α-D-glucose, D-fructose, D-galactose (weak), Inosine (weak), D-sorbitol, D-mannitol, D-arabitol, myo-inosiyol (weak), glycerol L-alanine, L-arginine (weak), L-glutamic acid, L-pyroglutamic acid (weak), L-serine, pectin, D-gluconic acid, methyl pyruvate, L-lactic acid, D/L-malic acid, bromo-succinic acid, propionic acid, acetic acid, γ-hydroxy-butyric acid, β-hydroxy-D,L butyric acid and formic acid (weak) in the Biolog GEN III MicroPlate system. Other detailed biochemical and physiological characteristics of the strain are given in Table 1. Strain G5-11^T and its reference strain were resistant to rifampicin (10 µg·mL⁻¹), tetracycline (25 µg·mL⁻¹), spectinomycin (50 µg·mL⁻¹) and Nalidixic acid (30 µg·mL⁻¹) and sensitive to streptomycin (50 µg·mL⁻¹), gentamicin (40 µg·mL⁻¹), erythromycin (20 µg·mL⁻¹) and chloramphenicol (10 µg·mL⁻¹). Strain G5-11^T was also sensitive to ampicillin (100 µg·mL⁻¹) (table S1).

Table 1

Differences in phenotypic characteristics of strain G5-11^T and the other type strains in genus *Halomonas*.
Characteristic	1	2	3^a	4^b
Colonial morphology and pigmentation	creamy and smooth	creamy and smooth	cream and round colonies	white, opaque colonies
growth pH	6.0-9.0 (8.0)	6.0-9.0 (8.0)	4.0-10.0 (8.0)	5.0-9.0 (8.0)
growth temperature (℃)	4-35 (30)	4-37 (30)	4-37 (30)	4-45 (30/37)
growth NaCl( %, w/v)	3.0-15.0 (5.0)	2.0-25.0 (5.0)	3.0–25.0 (12.0-15.0)	3.5–20.0 (3.5-8.0)
DNA G+C content (mol %)	61.0	65.0	60.8	60.5±0.5
facultatively anaerobic growth	+	-	+	+
Nitrate reduction	+	w	+	+
indole production	-	-	-	+
Activities of
urease	+	+	-	+
trypsin	-	-	+	-
Hydrolysis of
casein	+	+	+	-
gelatin	-	-	-	+
aesculin	-	-	-	+
Utilization of
lactose	-	+	-	+
D-mannitol	+	+	-	+
D-mannose	-	w	-	+
D-cellobiose	-	w	+	+
Biolog GENIII
Acetoacetic Acid	-	+	NT	NT
Tween 40	-	+	NT	NT
Inosine	w	-	NT	NT
D-Lactic Acid Methyl Estetr	-	+	NT	NT
Strains: 1, G5-11^T; 2, H. taeanensis DSM 16463^T; 3, H. niordiana LMG 31227^T 4, H. elongata ATCC33173^T.
All data were from this study unless otherwise indicated.
+, Positive; -, negative; w, weakly positive. NT, not test. ND, not determined or data not available in relevant literature.
^aData from Dieguez et al. (2020)
^bData from VREELAND et al. (1980b)
All strains of the genus Halomonas were Gram-staining-negative, motile (by means of several flagella), halophilic, catalase-positive and had the ability to reduce nitrate and ferment glucose. All strains could utilize glycerol, D-glucose, sucrose and none could hydrolysis of starch, tween 80 and D-salicin. All strains were negative for β-Galactosidase.

Phylogenetic and genotypic characteristics

Based on the results from EzBioCloud server, the closest relatives of the strain G5-11^T were members of the class Gammaproteobacteria and family Halomonadaceae, with the highest 16S rRNA gene sequence similarity of 98.3 % with H. niordiana LMG 31227^T (SDSD01000014) and H. taeanensis DSM 16463^T (AY671975). The 16S rRNA gene sequence phylogenetic tree showed that strain G5-11^T shared the same position with H. niordiana LMG 31227^T and H. taeanensis DSM 16463^T (Fig. S1). The same relationship was also found in trees reconstructed using 23S rRNA (Fig. S2) and using a set of 92 UBCGs (up-to-date bacterial core genes) based on the whole genome (Fig. 2), which importantly provides support that strain G5-11^T should be recognized as a novel species within the genus Halomonas.

Strain G5-11^T possessed a genome of 3395587 bp, comprising 3084 predicted genes. The draft genome of strain G5-11^T consisted of 50 contigs, with an N50 value of 139373 bp and an N90 contig lenth of 54722 bp. The complete genome of strain G5-11^T had 100-fold depth of sequencing coverage. The percentages for nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) with respect to the related species of Halomonas is showed (Table 2). The average ANI values and dDDH between G5-11^T and the reference strains were all below the cut-off level (95-96 % and 70 %, respectively) for species delineation.

Table 2

Results of ANI and dDDH between genomes of *H. salinarum* G5-11^T and those most closely related species in the genus *Halomonas*.
Bacterial species	OrthoANIu value (%)			dDDH value (%)
Bacterial species	1	2	3	1	2	3
H salinarum G5-11^T	82.91	93.05	76.32	26.70	50.70	21.50
Strains: 1, H. taeanensis DSM 16463^T; 2, H. niordiana LMG 31227^T; 3, H. elongata DSM 2581^T
All data were obtained in this study

NR (Non-Redundant Protein Databas), a non-redundant protein database, created and maintained by NCBI, characterized by comprehensive species annotation content for use in taxonomy. The annotation result showed most amino acid sequences in G5-11^T were highly homologous with Halomonas. Of these, the most abundant annotated genes came from H. taeanensis (2243). Halophilic microorganisms are considered a resource for industrial catalysts that function in organic solvent, high salt or other extreme conditions that most organisms cannot tolerate (Adamiak et al. 2016). To confronte with the problem of passive permeation of H⁺ and Na⁺ through the cytoplasmic membrane, the Na⁺/H⁺ antiporters play pivotal roles in intracellular Na⁺ excretion, which are important for these microbes salt stress resistance (Mesbah et al. 2009), and the vast majority of prokaryotes cope with increasing osmolarity also by uptake or synthesis of compatible solutes (Averhoff and Müller 2010). NhaD is an antiporter known so far only from halophilic or haloalkaliphilic Proteobacteria. This type of antiporter provides a special mechanism for adaptation to hypersaline habitats (Kurz et al. 2006). The annotation result showed the NhaD in G5-11^T shared the highest similarity with this of H. elongata (Identity value 76.4 %) suggest this Na⁺: H⁺ antiporter may contribute to sodium tolerance and pH homeostasis of G5-11^T. Multicomponent Na⁺:H⁺ antiporter, NhaA-G, which have alkaline pH optima and apparent K_m values for Na⁺ that are among the lowest reported for bacterial Na⁺/H⁺ antiporters, were also found in G5-11^T, supported that the strain G5-11^T exhibit considerable salt-tolerance and pH stability. Morever, swissprot annotation analysis showed that several possible genes for the biosynthesis of the compatible solute ectoine (e.g.,ectA, ectB, ectC) were found (table S2). A schematic diagram of the ect gene clusters in the genus H.taeanensis, H.niordiana and H.elongata was shown in Fig. S3. The combination of these strategies allowed strain G5-11^T grow over a range of extracellular pH and Na⁺ concentrations and protect biomolecules under multiple extreme conditions.

Chemotaxonomic characteristics

The isoprenoid quinone of strain G5-11^T was Q-9, the same as for the phylogenetically closely related strains H. taeanensis 16463^T (Lee et al. 2005). The major fatty acids in strain G5-11^T were summed feature 8 (C_18:1ω7c/ C_18:1ω6c, 32.4 %) and C_16:0 (24.1 %), followed by summed feature 3 (C_16:1ω7c/ C_16:1ω6c, 23.9 %), a pattern consistent with other members of genus Halomonas (Table S3). The polar lipid profile consists of PC, PG, DPG, PE and UAL as the major components (Fig. S4). On the basis of phylogenetic, physiological and chemotaxonomic analyses strain G5-11^T represents a novel species of the genus Halomonas.

Taxonomic conclusions

Phylogenetic analysis and chemotaxonomic characteristics, including isoprenoid quinone, major cellular fatty acid and DNA G+C content, unequivocally supported the placement of strain G5-11^T within the genus Halomonas. However, differences in phenotypic characteristics between strain G5-11^T and related type strains suggested that strain G5-11^T could be differentiated from this single recognized species of the genus Halomonas. Therefore, based on the molecular and physiological characteristics of strain G5-11^T, it is proposed that this strain represents a novel species, Halomonas salinarum sp.nov.

Description of Halomonas salinarum sp.nov

Halomonas salinarum (sa.li.na'rum. L. gen. pl. n. salinarum of salt works)

Cells are Gram-staining-negative, facultatively aerobic, moderately halophilic, rod-shaped, motile, 1.2-2.1 µm long and 0.7-1.1 µm wide. Colonies are creamy white to pale yellow. Growth occurs on 4–35 ℃ (optimum, 30 ℃), at pH 6.0-9.0 (optimum, 8.0) and with 3-15 % NaCl (optimum, 5 %). Growth occurs on Gibbson, MA, LB and R₂A media with 5 % NaCl. Acid is produced from D-Ribose (weak), D-Tagatose (weak) and Potassium 5-KetoGluconate (API 50CHB). Positive for catalase and oxidase. Positive for alkaline phosphatase, esterase lipase (C8) (weak), leucine aramidase, valine aramidase, naphthol-AS-BI-phosphohydrolase and α-glucosidase (API ZYM). In API 20NE tests, reduction of nitrate, urease, arginine dihydrolase (weak) and assimilation of D-glucose, L-arabinose, D-mannitol, D-maltose and phenylacetic acid are positive. Negative for production of indole and hydrolysis of gelatin. Isoprenoid quinones was Q-9, and the most abundant fatty acid were summed feature 8 (C_18:1ω7c/ C_18:1ω6c) followed by C_16:0 and summed feature 3 (C_16:1ω7c/ C_16:1ω6c). The polar lipid profile of the isolates was found to consist of PC, PG, DPG, PE and UAL as the major components. The DNA G+C content of this type of strain is 61.0 mol%.The type strain is G5-11^T (=CGMCC 1.12051^T=LMG 31677^T), which was isolated from saline soil collected in Yingkou, Liaoning, PR China.

The type strain, G5-11^T (=CGMCC 1.12051^T=LMG 31677^T), was isolated in the process of investigating the microbial diversity of saline soil from Yingkou, Liaoning, PR China. The G+C content of genomic DNA is 61.0 mol%. The GenBank accession number for the 16S rRNA gene sequence of strain G5-11^T is JQ010842. The GenBank accession number for the 23S rRNA gene sequence of strain G5-11^T is MT901368. The whole genome of strain G5-11^T has deposited at DDBJ/ENA/GenBank under the accession number WWNB00000000.

DPG	Diphosphatidylglycerol
PE	Phosphatidylethanolamine
PG	Phosphatidylglycerol
PC	Phosphatidylcholine
UAL	Unidentified aminolipid

Acknowledgments The authors are grateful to Professor Jesús L.Romalde (Departamento de Microbiología y Parasitología, CIBUS-Facultad de Biología, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain) who kindly provide fatty acid data of H. niordiana LMG 31227^T.

Author’s contributions YLY designed research and project outline. YLY and FLL performed isolation, deposition and polyphasic taxonomy. YLY performed genome analysis. YLY and FFL drafted the manuscript. LW revised the manuscript. All authors read and approved the final manuscript.

Funding information This work was supported by the National Natural Science Foundation of China (Grant No. 41977198).

Conflicts of interest All authors declare that there is no conflict of interest in this article.

Ethical statement No experiments with humans or animals were carried out.

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Halomonas Salinarum sp. nov., a Moderately Halophilic Bacterium, Isolated from Saline Soil in Yingkou, China

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