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Article

Taxogenomic and Metabolic Insights into Marinobacterium ramblicola sp. nov., a New Slightly Halophilic Bacterium Isolated from Rambla Salada, Murcia

by
Ana Durán-Viseras
1,2,*,
David J. Castro
1,2,
José Carlos Reina
1,2,
Victoria Béjar
1,2 and
Fernando Martínez-Checa
1,2,*
1
Microbial Exopolysaccharide Research Group, Department of Microbiology, Pharmacy Faculty, Campus de Cartuja s.n., 18071 Granada, Spain
2
Biomedical Research Center, Institute of Biotechnology, 18016 Granada, Spain
*
Authors to whom correspondence should be addressed.
Microorganisms 2021, 9(8), 1654; https://doi.org/10.3390/microorganisms9081654
Submission received: 29 June 2021 / Revised: 28 July 2021 / Accepted: 29 July 2021 / Published: 3 August 2021
(This article belongs to the Special Issue Halophilic Microorganisms)
Browse Figures
Versions Notes

Abstract

:
A Gram-negative, motile, rod-shaped bacteria, designated D7T, was isolated by using the dilution-to-extinction method, from a soil sample taken from Rambla Salada (Murcia, Spain). Growth of strain D7T was observed at 15–40 °C (optimum, 37 °C), pH 5–9 (optimum, 7) and 0–7.5% (w/v) NaCl (optimum, 3%). It is facultatively anaerobic. Phylogenetic analysis based on 16S rRNA gene sequence showed it belongs to the genus Marinobacterium. The in silico DDH and ANI against closest Marinobacterium relatives support its placement as a new species within this genus. The major fatty acids of strain D7T were C16:0, summed feature 3 (C16:1 ω7c/C16:1 ω6c) and summed feature 8 (C18:1 ω7c/C18:1 ω6c). The polar lipid profile consists of phosphatidylethanolamine, phosphatidylglycerol and two uncharacterized lipids. Ubiquinone 8 was the unique isoprenoid quinone detected. The DNA G + C content was 59.2 mol%. On the basis of the phylogenetic, phenotypic, chemotaxonomic and genomic characterization, strain D7T (= CECT 9818T = LMG 31312T) represents a novel species of the genus Marinobacterium for which the name Marinobacterium ramblicola sp. nov. is proposed. Genome-based metabolic reconstructions of strain D7T suggested a heterotrophic and chemolitotrophic lifestyle, as well as the capacity to biosynthetize and catabolize compatible solutes, and to degrade hydrocarbon aromatic compounds.

1. Introduction

The genus Marinobacterium belongs to the family Alteromonadaceae, order Alteromonadales, class Gammaproteobacteria. At the time of writing, the genus Marinobacterium comprises 18 different species, from which the type species is Marinobacterium georgiense, a later heterotypic synonym of Marinobacterium iners [1,2].
Cells of members of this genus are Gram-stain-negative rods, motile by a single or two polar flagella [3]. They are slightly halophilic microorganisms, requiring NaCl for growth [3]. Some members are able to grow on aromatic compounds or to fixate nitrogen under limiting oxygen conditions [3]. Major fatty acids include C16:0, C16:1 ω6c and/or C16:1 ω7c, and C18:1 ω6c and/or C18:1 ω7c. Ubiquinone (Q-8) is the major respiratory quinone [3].
Species of the genus Marinobacterium have been isolated from a variety of ecological niches including marine habitats, such as tidal flats [4,5,6], marine sediments [7,8,9], seawater [10,11,12,13,14,15], estuarine sediments [16], marine organisms [17], coastal tidal-flat plant rhizospheres [18,19] and pulp mill waste [1].
Using culture-independent techniques, members of the genus Marinobacterium were also detected in a wide range of habitats including soil samples from tidal freshwater wetlands in China [20], from mangrove river sediment in Taiwan [21], surface sediments from the deep eastern Mediterranean Sea [22] and from the Cochin estuary in the southeastern Arabian Sea [23], seawater samples from the Upper Gulf of Thailand [24], in sodium saccharin wastewater [25], and in different oil or petroleum reservoirs from Brazil, Malaysia and China [26,27,28,29], among others.
During the course of a study of a soil sample collected from Rambla Salada, by using the dilution-to-extinction isolation methodology, strain D7T was isolated in pure culture. This technique allows us to separate individual bacterial cells to initiate enrichment cultures, improving the recovery of slow-growing microorganisms or microorganisms that are apparently uncultivable [30].
Rambla Salada is a hypersaline habitat located in the Province of Murcia (southeastern Spain). It was declared as an area with special interest by the European Union, and as a protected wildfowl zone by the regional government of Murcia (BORM 10 September 1998). The salinity of this protected habitat is mainly due to the presence of Na+, Cl−, SO42− and Ca2+ ions. It is an extensive area formed by sedimentary materials in which, due to the low rainfall together with underground water that emerges, originates a number of streams and wadis. The prokaryotic community of Rambla Salada has been studied by our research group during recent decades [30,31,32,33], and we have described so far six novel halophilic bacterial species from this athalassohaline habitat: Idiomarina ramblicola [34], Halomonas cerina [35], Halomonas ramblicola [36], Blastomonas quesadae [37], Roseovarius ramblicola [38] and more recently, Roseovarius bejariae [39].
In the publication at hand, we describe the isolation and taxogenomic characterization of strain D7T, based on a polyphasic approach including genomic data. Results confirm it is a new species within the genus Marinobacterium, for which we propose the name of Marinobacterium ramblicola sp. nov. Moreover, based on a detailed genomic study, the metabolism of this new taxon was studied in depth.

2. Materials and Methods

2.1. Bacterial Strains

Strain D7T was isolated in this study from a soil sample taken from Rambla Salada, a hypersaline steep-sided river (rambla), located in the Province of Murcia, southeast Spain, 38°07′27.1″ N 1°07′01.4″ W. The physicochemical parameters of pH, oxygen (mg L−1) and salinity (g L−1) of the sampling location in Rambla Salada were 6.3, 10.2 and 44.4, respectively.
The sample was collected using a sterile polycarbonate tube, taken immediately to the laboratory and stored at 4 °C until study. The pH of the sample was close to neutral and salinity was around 40 g L−1. For the isolation, we used S3 medium, a low-nutrient medium [40] supplemented with 3% (w/v) sea-salt solution [41] and the dilution-to- extinction approach as a cultivation method, described previously by Castro et al. [38]. This approach is a technique that improves the isolation of slow-growing species or apparently uncultivable species [42,43,44]. The extinction cultures were incubated at 25 °C for 30 days and after, the contents of wells were then re-isolated on Reasoner’s 2A (R2A) medium plates [45]. The isolated strain was maintained and routinely grown in R2A with 3% (w/v) sea-salt solution at 30 °C as well as on marine agar (MA; 2216, Difco, Sparks, MD, USA).
For taxonomic comparison purposes, Marinobacterium zhoushanense KCTC 42782T was used in this study and routinely grown in the same media as strain D7T.

2.2. DNA Extraction, Purification and Sequencing

Genomic DNA was extracted using an X-DNA purification kit (Xtrem Biotech, Granada, Spain) from an overnight culture of strain D7T in R2A medium. The 16S rRNA gene was amplified by PCR using the universal primers for bacteria 16F27 and 16R1488 [46]. The obtained PCR product was then purified, cloned into the pGEM-T vector (Promega, Madison, WI, USA), and sequenced by direct sequencing using the ABI prism dye-terminator, cycle-sequencing ready-reaction kit and the ABI prism 377 sequencer according to PerkinElmer’s instructions. The GenBank/EMBL/DDBJ accession number for the sequence of the 16S rRNA gene is MG773714. The genome of strain D7T was sequenced by using the Illumina MiSeq methodology (PE 150 × 2).

2.3. Phylogenetic Analysis Based on 16S rRNA Gene Sequence Comparison

Phylogenetic analyses based on the 16S rRNA gene were conducted as described previously [38,47]. The phylogenetic neighbors’ identification and the pairwise 16S rRNA gene sequence similarities calculations were carried out by using the EzBiocloud server “www.ezbiocloud.net” [48] (accessed on April 2021). Phylogenetic and molecular evolutionary analyses were conducted using Mega v. 7 [49]. Clustering was determined using the neighbor-joining and maximum-likelihood algorithms and the evolutionary distances were computed using the Jukes–Cantor method [50]. The analysis involved 34 nucleotide sequences and the stability of the clusters was determined by a bootstrap analysis (1000 replications).

2.4. Genomic Analyses

Illumina reads of strain D7T were trimmed using a combination of software tools implemented in the BBMap project [51], and de novo assembled using SPADES v3.11.1 [52]. CheckM v1.0.18 [53] and Quast v5.0.2 [54] were used for assembly quality checks. The genome of strain D7T was deposited in GenBank/EMBL/DDBJ under the accession number JAHREP000000000.
The genome of strain D7T was annotated using BlastKOALA [55] and metabolic pathways were analyzed using KEGG [56].
Average Nucleotide Identity (ANI) and in silico DNA–DNA hybridization (DDH) values were calculated using the OrthoANI-usearch (OrthoANIu) software [57] and the Genome-to-Genome Distance Calculator (GGDC) website [58] with formula 2 [59], respectively.
InteractiVenn software [60] was used to display the Venn diagram.

2.5. Phylogenomic Reconstruction

Predicted protein sequences were compared using an all-versus-all BLAST search [61]. A total of 134 proteins were shared between all studied genomes and aligned using MUSCLE v3.8.31 [62]. The concatenated and aligned orthologous genes were used to build the phylogenomic tree in Mega v. 7 [49].

2.6. Phenotypic Characterization

Phenotypic analysis was conducted in order to characterize strain D7T. The optimum and the range of salt growth conditions for strain D7T were evaluated in R2A medium to which were added different NaCl concentrations: 0, 0.5, 1, 3, 5, 7.5, 10, 15, 20, 25 and 30% (w/v). The optimal (and range) growth pH of D7T was evaluated by growing the strain under different pH values (4, 5, 6, 7, 8, 9, 10 and 11). These pH values were reached using the following buffer systems: 0.1 M citric acid/0.1 M sodium citrate (pH 4.0–5.0); 0.1 M KH2PO4/0.1 M NaOH (pH 6.0–8.0); 0.1 M NaHCO3/0.1 M Na2CO3 (pH 9.0–10.0); and 0.2 M KH2PO4/0.1 M NaOH (pH 11.0) [63]. The effect of different temperatures (0, 5, 15, 20, 25, 28, 30, 32, 35, 37, 40 and 45 °C) was also assessed using marine broth (MB; Difco, Sparks, MD, USA).
Gram staining was performed according to the method described by Komagata [64]. Growth under anaerobic conditions was determined in an anaerobic jar using AnaeroGen (Oxoid) and an anaerobic indicator (Oxoid, Hampshire, UK) using marine agar (MA; Difco). Motility was observed using log-phase culture according to the hanging-drop method [65]. Oxidase activity was determined with 1% (v/v) tetramethyl-p-phenylenediamine [66] and catalase activity was examined by bubble production with 3% (v/v) H2O2 solution [65]. The reduction of nitrate and nitrite and gas production were detected by adding the Griess-Ilosvay’s reagent (Merck) in cultures grown in peptone broth supplemented with 1% KNO3. Other biochemical character, carbon utilization, sugar fermentation and enzymatic tests were carried out by using the GEN III MicroPlateTM system (Biolog), API 20NE, API 50CH and API ZYM strips (bioMerieux, Marcy I’Etoile, France) according to the manufacturers’ instructions.
Scanning electron microscope images of strain D7T were produced on an FIB-FESEM (CrossBeam NVision 40, Carl Zeiss SMT) to determine bacterium size and type of flagella.

2.7. Chemotaxonomic Characterization

The fatty acids of strain D7T were analyzed at the Spanish Type Culture Collection (CECT). Cells were grown on MA for 48 h, incubated at 30 °C. The whole-cell composition of the fatty acids was determined by GC using the midi microbial identification system [67]. The fatty acid profile was obtained with an Agilent 6850 gas chromatograph using the database TSBA6 145 [68].
Analysis of polar lipids and respiratory quinones of strain D7T was carried out by the Identification Service of DSMZ, Braunschweig, Germany. Polar lipids were extracted following the protocol described by Bligh and Dyer [69]. Polar lipids were separated by two-dimensional silica-gel thin-layer chromatography (Macherey-Nagel Art. No. 818135) following the protocol described by Tindall et al. [70]. The two-stage method described by Tindall [71,72] was used to first extract respiratory lipoquinones followed by polar lipids.

3. Results and Discussion

3.1. Phylogenetic Analysis

During the course of the study of a soil sample from Rambla Salada (38°07′27.1″ N 1°07′01.4″ W), a hypersaline river located in Murcia (southeast of Spain), a novel strain designated D7T was isolated and selected for further studies.
Based on the 16S rRNA gene sequence analysis, strain D7T (1490 bp) was most closely related to the genus Marinobacterium, exhibiting the highest 16S rRNA gene sequence similarity to Marinobacterium zhoushanense WM3T (98.0%), followed by Marinobacterium lutimaris DSM 22012T (95.7%), Marinobacterium mangrovicola Gal22T (95.3%) and Marinobacterium litorale IMCC 1877T (95.1%). Moreover, the 16S rRNA gene sequence similarities to other genera, such as Neptunomonas and Nitrincola, were always equal or lower than 93.5%. The phylogenetic analysis based on the multiple sequence alignment of the 16S rRNA gene using the neighbor-joining algorithm (Figure 1), indicated that strain D7T belongs to the genus Marinobacterium clustering with Marinobacterium zhoushanense WM3T but was located in an independent branch with a high bootstrap value (Figure 1), indicating that the new strain could represent a new member of the genus Marinobacterium. A phylogenetic tree devised using the maximum-likelihood algorithm exhibited similar topologies.

3.2. Genomic Characteristics

The draft genome sequence of strain D7T was obtained and compared with that of the closest phylogenetic species, Marinobacterium zhoushanense CGMCC 1.15341T, and with those of other members of the genus Marinobacterium with available genomes (Table 1). The draft genome of strain D7T was de novo assembled into a total of 69 contigs, with a N50 value of 150,886 bp, a sequencing depth of 844X and a completeness of 99.9%. This genome sequence is in accordance with the minimal standards for the use of genome data for the taxonomy of prokaryotes [73]. The G+C content and genome size of strain D7T were 59.2 mol% and 4,897,523 bp, respectively; those values were within the range of the genomes of the genus Marinobacterium, which ranged from 54.9 to 62.1 mol%, and from 3,653,655 to 5,637,742 bp, respectively (Table 1). Additional genomic characteristics are detailed in Table 1. Besides, the 16S rRNA gene sequence of strain D7T obtained from the draft genome sequence was identical to that from the PCR, verifying the authenticity of this genome.
In addition, a Venn diagram displaying the number of genes shared between strain D7T, Marinobacterium zhoushanense CGMCC 1.15341T and Marinobacterium lutimaris DSM 22012T was obtained (Figure 2). A total of 645 genes were shared between M. zhoushanense CGMCC 1.15341T, M. lutimaris DSM 22012T and strain D7T, while 462 genes were shared between M. lutimaris DSM 22012T and strain D7T, and 561 genes between M. zhoushanense CGMCC 1.15341T and strain D7T (Figure 2). A total of 2780 genes were identified as unique to strain D7T (Figure 2). These results indicate that strain D7T was unique from its closely related species.

3.3. Phylogenomic Analysis

According to the minimal standards for the use of genome data for the taxonomy of prokaryotes [73] and to confirm the phylogenomic relationships previously obtained by 16S rRNA gene sequence comparison, a phylogenomic tree based on core orthologous translated genes from strain D7T and closely related members of the genus Marinobacterium was also obtained. A total of 134 single-copy orthologous genes were shared between all studied genomes and the phylogenomic tree reconstruction (Figure 3), clearly reflects that strain D7T constitutes a monophyletic clade distinct from any other previously described species of the genus Marinobacterium, and therefore, supporting the placement of strain D7T as a new species within this genus.

3.4. Average Nucleotide Identity (ANI) and In Silico DNA–DNA Hybridization (DDH)

To elucidate if strain D7T may constitute a new species within the genus Marinobacterium, the genome-based sequence similarity analysis (ANI and in silico DDH) between strain D7T and members of this genus was performed. For species delineation, the proposed and accepted boundaries for ANI and DDH are 95–96% and 70%, respectively [58,74,75].
The ANI and DDH values between strain D7T and Marinobacterium zhoushanense CGMCC 1.15341T, the closest phylogenetic neighbor, were 86.7% and 31.3%, respectively (Figure 4). In addition, the ANI and DDH estimations of strain D7T in comparison to those of the other members of the genus Marinobacterium with available genomes were in all cases lower than the established cutoff values (Figure 4). These results support the conclusion that strain D7T represents a novel species of the genus Marinobacterium.

3.5. Chemotaxonomic Characterization

To taxonomically describe strain D7T as a new species, the complete chemotaxonomic characterization of this strain was performed. The polar lipid profile of strain D7T includes phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and two uncharacterized lipids (L) (Figure S1).
The fatty acids composition of strain D7T and the type strains of related species of the genus Marinobacterium are shown in Table 2. In accordance with members of the genus Marinobacterium [3], the major cellular fatty acids (>10%) of strain D7T were C16:0 (28.7%), summed feature 3 (C16:1 ω7c/C16:1 ω6c) (26.6%) and summed feature 8 (C18:1 ω7c/C18:1 ω6c) (25.2%). The fatty acid profile of strain D7T was similar to those of the reference strains, but differing in their proportion (Table 2); hence, reaffirming its condition as a different species.
The respiratory quinone of strain D7T was ubiquinone-8 (Q-8), which is consistent with the rest of the members of the genus Marinobacterium [3].

3.6. Phenotypic Characterization

The phenotypic characteristics of strain D7T were described and compared with those of Marinobacterium zhoushanense KCTC 42782T. Cells were Gram-staining-negative, short motile rods (0.5–0.6 × 1.0–1.8 µm) with a single polar flagellum (Figure S2). They were catalase and oxidase positive. Nitrate and nitrite were reduced. Glucose was not fermented. Weak growth was observed under anaerobic conditions. When tested on MA, growth of strain D7T was observed at 15–40 °C (optimum, 37 °C), pH 5–9 (optimum, 7) and 0–7.5% (w/v) NaCl (optimum, 3%). Other characteristics of strain D7T are given in the species description and those that differ from the strain type of the closest related species of the genus Marinobacterium are shown in Table 3.

3.7. Metabolism of Strain D7T

Metabolic insights after the in-depth genomic analysis of strain D7T suggest a heterotrophic and chemolithotrophic lifestyle for this strain. In relation to its heterotrophic capabilities, central carbohydrate pathways such as glycolysis, gluconeogenesis, pentose phosphate, Entner–Doudoroff, tricarboxylic acid and glyoxylate cycle were detected (Figure 5). For pyruvate oxidation to acetyl-CoA, genes encoding pyruvate dehydrogenase (aerobic route) and pyruvate ferredoxin oxidoreductase (anaerobic route) were present. On the other side, a large number of ABC transporters for carbohydrate uptake (i.e., multiple sugar, glucose/mannose or fructose) were also identified in the studied genome (Figure 5).
As part of the nitrogen metabolism of strain D7T, complete pathways for nitrogen fixation, dissimilatory nitrate reduction and denitrification were detected on its genome (Figure 5). While genomic evidence for nitrogen fixation and nitrate reduction to ammonia via dissimilatory nitrate reduction pathway has been previously suggested for other Marinobacterium species [3], the whole set of genes encoding all steps of the denitrification was not identified before in any other members of this group [3]. Only Marinobacterium jannaschii exhibited the almost complete route, but lacked the key enzyme (NirK) [3]. Moreover, in the specific case of strain D7T, the nitrate reduction capacity of this strain was also detected during the nitrate reduction phenotypic test. Several transporters for exogenous nitrogen-rich organic compounds uptake, such as Amt for ammonia, ABC for amino acids, putrescine or urea, with others, were found (Figure 5). In addition, various complete aminoacidic biosynthetic routes (i.e., serine, threonine, cysteine, valine/isoleucine, leucine, lysine, ornithine, arginine, proline, histidine and tryptophan) were encountered in this genome.
Like many halotolerant microorganisms, to cope with osmotically varying conditions, D7T encode genes for the synthesis and uptake of different compatible solutes. Glycine betaine is one of the most important osmoprotectants in prokaryotes that could also serve as an energy and carbon source in hypersaline environments [76]. The key enzymes choline dehydrogenase (BetA) and glycine betaine aldehyde dehydrogenase (BetB), involved in the biosynthesis of glycine betaine from choline, were recognized during the genomic analysis of strain D7T (Figure 5). The gene clusters gbcAB, dgcAB and soxBDAG for its further catabolism to glycine were also identified. This strain also possesses transporters for the uptake of this compound, such as the glycine betaine BCCT family and ABC transporters (Figure 5). No evidence for the alternative route for glycine betaine biosynthesis from glycine (via glycine and sarcosine methyltransferase and dimethylglycine methyltransferase) was identified in the studied genome.
Ectoine is another osmotic solute widely synthetized by halophilic bacteria [76]. The presence of the complete biosynthesis pathway in this strain suggests its capacity to additionally synthetize ectoine as a compatible solute (Figure 5). Besides, the ectoine hydroxylase (EctD) enzyme and the doeBDAC gene cluster, coding for Nα-acetyl-L-2,4-diaminobutyrate deacetylase (DoeB), diaminobutyrate transaminase (DoeD), ectoine hydrolase (DoeA) and aspartate semialdehyde dehydrogenase (DoeC), were also identified in this genome, indicating the ability to likewise synthetize its hydroxylated derivate, 5-hydroxyectoine, and to degrade ectoine, respectively (Figure 5). Considering ectoine biosynthesis is energetically more expensive than the betaine one, it would be reasonable to believe that this strain only synthetizes ectoine under starving betaine or choline concentrations.
In addition, other several transporters related to osmotic stress for potassium uptake and sodium extrusion were also found in the genome.
Entire pathways for the catabolism of aromatic hydrocarbons (such as benzoate, benzene and anthranilate) to catechol were encountered in the genome of strain D7T, fueling the catechol meta-cleavage pathway for its further breaking down (Figure 5). In the same way, other Marinobacterium representatives (i.e., M. aestuarii, M. stanieri, M. profundum and/or M. jannaschii) were predicted to degrade benzoate or benzene, although in some cases through the ortho-cleavage pathway [3,16].
Noteworthily, strain D7T encodes a sulfide:quinone oxidoreductase (SQR) which is predicted to oxidize sulfide to elemental sulfur, and thus exhibits a potential chemolithotrophic energetic metabolism for this strain. The SQR enzyme was also previously identified in the genomes of M. jannaschii and M. litorale [3]. Several others sulfur-based lithotrophy genes were also encoded by diverse Marinobacterium species [3]. On the other hand, the assimilatory sulfate reduction pathway via 3′-phosphoadenosine-5′-phosphosulfate (PAPS) was identified in the genome of strain D7T (Figure 5), reflecting its capacity to reduce sulfate to sulfide with the aim of satisfying sulfur nutritional requirements.
Acetate is a major product of the metabolism formed during fermentation by many bacteria and other organisms [77]. In strain D7T, acetate is produced from Acetyl-CoA catalyzed by the phosphate acetyltransferase (Pta) and acetate kinase (AckA) enzymes (Figure 5). In addition to acetate, this reaction generates ATP, hence contributing to the energy metabolism of the cell.
Finally, consistently with its microscopical visualization, genes encoding flagellum were found during the genomic analysis.

4. Conclusions

As a conclusion of the polyphasic taxogenomic analyses performed in this study, strain D7T represents a new species within the genus Marinobacterium, for which the name Marinobacterium ramblicola sp. nov. is proposed. We enclose below the taxonomic description of this new species. The detailed genomic analysis of Marinobacterium ramblicola D7T inferred a versatile energetic metabolism for this new taxon, characterized by a typical aerobic electron transport chain, oxidation of sulfur compounds, and nitrogen assimilation and fixation pathways. Likewise, based on the genomic inspection, it was suggested that the new species has the ability to biosynthetize or catabolize compatible solutes (i.e., ectoine or glycine betaine) and to degrade several aromatic hydrocarbons (i.e., benzene, benzoate and anthranilate).

Description of Marinobacterium ramblicola sp. nov.

Marinobacterium ramblicola [ram.bli’co.la. Spanish fem. n. rambla sandy ground; L. suff. -cola (from L. masc. or fem. n. incola) inhabitant; N.L. n. ramblicola inhabitant of a rambla].
Cells are motile Gram-stain-negative rods (0.5–0.6 × 1.0–1.8 µm). Colonies are 0.5 mm in size, pale yellow, circular, convex and opaque when grown on MA medium. Catalase and oxidase positive. Growth occurs at 15–40 °C (optimum, 37 °C), at pH 5–9 (optimum, 7) and with 0–7.5% (w/v) NaCl (optimum, 3%). Facultatively anaerobic. Cells are positive for nitrate reduction without gas formation. Indol is not produced. Gelatin and urea are not hydrolyzed. In Biolog GEN III MicroPlatesTM, cells are positive for the assimilation of L-alanine, D-aspartic acid, L-aspartic acid, dextrin, D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, L-histidine, L-arginine, D-turanose, α-D-glucose, D-fructose, glycerol, glycyl-L-proline, L-glutamic acid, L-pyroglutamic acid, L-serine, pectin, D-galacturonic acid, L-galactonic acid lactone, acetic acid, acetoacetic acid, D-gluconic acid, quinic acid, p-hydroxy-phenylacetic acid, methyl pyruvate, D-lactic acid methyl ester, L-lactic acid, citric acid, α-hydroxy-butyric acid, α-keto glutaric acid, α-keto butyric acid, L-malic acid, β-hydroxy-D,L-butyric acid, bromo-succinic acid, Tween 40, γ-amino butyric acid, propionic acid, formic acid and sodium butyrate. Other organic substrates included in Biolog GEN III microplates are not utilized. Acids are produced from D-maltose and D-melezitose, but not from adonitol, D-arabinose, arbutin, D-arabitol, L-arabitol, D-cellobiose, D-fructose, D-glucose, glycerol, D-galactose, dulcitol, erythritol, L-lactose, D-mannose, D-ribose, D-xylose, L-xylose, methyl-β-D-xylopyranoside, L-sorbose, L-rhamnose, inositol, D-mannitol, D-sorbitol, methyl- α-D-glucopyranoside, methyl-α-D-mannopyranoside, N-acetylglucosamine, amygdalin, inulin, salicin, D-mellibiose, D-sucrose, D-raffinose, D-lyxose, starch, glycogen, D-trehalose, xylitol, gentiobiose, D-turanose, D-tagatose, D-fucose, L-fucose, potassium gluconate, potassium 2-ketogluconate or potassium 5-ketogluconate.
Enzymatic activities such as acid phosphatase, alkaline phosphatase, valine arylamidase, leucine arylamidase and napthol-AS-BI-phosphohydrolase are positive, but the activities cystine arylamidase, α-chymotrypsin, esterase (C4), esterase lipase (C8), lipase (C14), α-glucosidase, β-glucosidase trypsin, α-galactosidase, β-galactosidase, β-glucuronidase, N-acetyl- β-glucosaminidase, α-mannosidase and α-fucosidase are negative.
The isoprenoid quinone is Q-8. The polar lipids profile consists of phosphatidylethanolamine, phosphatidylglycerol and two uncharacterized lipids. Major fatty acids are C16:0, summed feature 3 (C16:1 ω7c/C16:1 ω6c) and summed feature 8 (C18:1 ω7c/C18:1 ω6c).
The type strain is D7T (= CECT 9818T = LMG 31312T), isolated from a hypersaline river located in Murcia, Spain.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and for the draft genome are MG773714 and JAHREP000000000, respectively.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/microorganisms9081654/s1, Figure S1: Two-dimensional TLC of polar lipids from strain D7T; Figure S2: Electron micrographs of negatively stained preparation of cells of strain D7T.

Author Contributions

D.J.C. isolated the strain. D.J.C. and A.D.-V. made the phylogenetic and phenotypic studies. J.C.R. did the data curation. A.D.-V. performed the genomic analyses and produced the figures. V.B. and F.M.-C. designed the study. A.D.-V. and F.M.-C. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by a grant from the Dirección General de Investigación Científica y Técnica (CGL2011-25748), Spain.

Acknowledgments

D.J.C. wishes to thank MINECO (Ministerio de Economía y Competitividad) for his “Industrial Doctorate” contract. J.C.R. was supported by a FPU fellowship (FPU15/01717) from the Spanish Ministry of Education, Culture and Sports. We thank Mohamed Tassi Mzanzi for his expertise in the electron-microscope studies.

Conflicts of Interest

The authors declare no conflict of interest.

References

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Figure 1. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences, showing the relationships of strain D7T and related species. Sequences’ accession numbers are shown in parentheses. Bootstrap values based on 1000 replicates are listed as percentages at branching points; only values >50% are shown. Filled circles represent common nodes recovered in the maximum-likelihood algorithm representation. Escherichia coli JCM 1649T was used as an outgroup. Bar, 0.02 substitutions per nucleotide position.
Figure 1. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences, showing the relationships of strain D7T and related species. Sequences’ accession numbers are shown in parentheses. Bootstrap values based on 1000 replicates are listed as percentages at branching points; only values >50% are shown. Filled circles represent common nodes recovered in the maximum-likelihood algorithm representation. Escherichia coli JCM 1649T was used as an outgroup. Bar, 0.02 substitutions per nucleotide position.
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Figure 2. Venn diagram showing the number of genes shared between the genome of strain D7T, Marinobacterium zhoushanense CGMCC 1.15341T and Marinobacterium lutimaris DSM 22012T.
Figure 2. Venn diagram showing the number of genes shared between the genome of strain D7T, Marinobacterium zhoushanense CGMCC 1.15341T and Marinobacterium lutimaris DSM 22012T.
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Figure 3. Neighbor-joining core gene phylogenetic tree including available genomes of the genus Marinobacterium and strain D7T. The tree was recovered from the alignment of 134 single-copy orthologous translated genes shared between these genomes. Bar, 0.1 substitutions per amino acid position.
Figure 3. Neighbor-joining core gene phylogenetic tree including available genomes of the genus Marinobacterium and strain D7T. The tree was recovered from the alignment of 134 single-copy orthologous translated genes shared between these genomes. Bar, 0.1 substitutions per amino acid position.
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Figure 4. Heatmap representing the Average Nucleotide Identity (ANI) and in silico DNA–DNA hybridization (DDH) percentages between strain D7T and related species of the genus Marinobacterium in which values are displayed by the colour key histograms on the upper panel. Strains: 1, Marinobacterium profundum PAMC 27536T; 2, Marinobacterium lutimaris DSM 22012T; 3, Marinobacterium litorale DSM 23545T; 4, Strain D7T; 5, Marinobacterium mangrovicola DSM 27697T; 6, Marinobacterium georgiense DSM 11526T; 7, Marinobacterium aestuarii ST58-10T; 8, Marinobacterium rhizophilum DSM 18822T; 9, Marinobacterium zhoushanense CGMCC 1.15341T; 10, Marinobacterium stanieri DSM 7027T; 11, Marinobacterium halophilum DSM 17586T; 12, Marinobacterium jannaschii DSM 6925T; 13, Marinobacterium nitratireducens CGMCC 1.7286T. Genome accession numbers are indicated in Table 1.
Figure 4. Heatmap representing the Average Nucleotide Identity (ANI) and in silico DNA–DNA hybridization (DDH) percentages between strain D7T and related species of the genus Marinobacterium in which values are displayed by the colour key histograms on the upper panel. Strains: 1, Marinobacterium profundum PAMC 27536T; 2, Marinobacterium lutimaris DSM 22012T; 3, Marinobacterium litorale DSM 23545T; 4, Strain D7T; 5, Marinobacterium mangrovicola DSM 27697T; 6, Marinobacterium georgiense DSM 11526T; 7, Marinobacterium aestuarii ST58-10T; 8, Marinobacterium rhizophilum DSM 18822T; 9, Marinobacterium zhoushanense CGMCC 1.15341T; 10, Marinobacterium stanieri DSM 7027T; 11, Marinobacterium halophilum DSM 17586T; 12, Marinobacterium jannaschii DSM 6925T; 13, Marinobacterium nitratireducens CGMCC 1.7286T. Genome accession numbers are indicated in Table 1.
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Figure 5. Metabolic reconstruction of strain D7T. Pathways included in color boxes from left to right: assimilatory sulfate reduction, acetate pathway, glycolysis, aromatic hydrocarbons’ degradation pathways, tricarboxilic acid cycle (TCA), ectoine biosynthesis and degradation pathways, nitrogen metabolism (dissimilatory nitrate reduction, denitrification and nitrogen fixation pathways), and glycine betaine biosynthesis and degradation pathways.
Figure 5. Metabolic reconstruction of strain D7T. Pathways included in color boxes from left to right: assimilatory sulfate reduction, acetate pathway, glycolysis, aromatic hydrocarbons’ degradation pathways, tricarboxilic acid cycle (TCA), ectoine biosynthesis and degradation pathways, nitrogen metabolism (dissimilatory nitrate reduction, denitrification and nitrogen fixation pathways), and glycine betaine biosynthesis and degradation pathways.
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Table
Table 1. General features of the genomes of strain D7T and other species of the genus Marinobacterium.
Table 1. General features of the genomes of strain D7T and other species of the genus Marinobacterium.
Feature12345678910111213
Size (bp)4,897,5234,734,3555,191,6083,925,2613,653,6555,174,2804,378,1725,568,3334,979,9475,546,8835,637,7425,360,5824,680,330
Contigs69291514747682115392266824
G+C (mol%)59.258.458.854.956.055.256.457.557.162.157.258.555.6
N50 (bp)150,886548,6475,191,608167,945130,350201,845137,897869,882922,765457,68061,011143,318388,460
Total genes4522440846143742344247054252513645174778505147684380
Protein coding genes4427428544613625332645694114502444144635486346044259
rRNA371883893ND3566
tRNA59738356716257696870826673
Accession numberJAHREP000000000BMIJ00000000CP015839FNRJ00000000PYGI00000000JHVJ00000000AUAZ00000000FNVQ00000000SMFU00000000BMLT00000000BCNS00000000ARJM00000000FTMN00000000
Strains: 1, Strain D7T; 2, Marinobacterium zhoushanense CGMCC 1.15341T; 3, Marinobacterium aestuarii ST58-10T; 4, Marinobacterium georgiense DSM 11526T; 5, Marinobacterium halophilum DSM 17586T; 6, Marinobacterium jannaschii DSM 6925T; 7, Marinobacterium litorale DSM 23545T; 8, Marinobacterium lutimaris DSM 22012T; 9, Marinobacterium mangrovicola DSM 27697T; 10, Marinobacterium nitratireducens CGMCC 1.7286T; 11, Marinobacterium profundum PAMC 27536T; 12, Marinobacterium rhizophilum DSM 18822T; 13, Marinobacterium stanieri DSM 7027T.
Table
Table 2. Fatty acid profile of strain D7T and closely related species of the genus Marinobacterium.
Table 2. Fatty acid profile of strain D7T and closely related species of the genus Marinobacterium.
Fatty Acids12 a3 a
Saturated:
C10:00.7--
C12:05.95.33.2
C14:00.8--
C16:028.722.022.8
C18:00.3TR1.3
Hydroxy:
C10:0 3-OH6.75.86.4
C12:0 2-OH--1.7
C16:0 3-OH0.3--
Cyclo:
C17:0 cyclo4.93.1TR
Summed features:
3 (C16:1 ω7c/C16:1 ω6c)26.629.516.9
8 (C18:1 ω7c/C18:1 ω6c)25.231.845.0
Strains: 1, Strain D7T; 2, Marinobacterium zhoushanense KCTC 42782T; 3, Marinobacterium lutimaris KACC 13703T. Cells of strain D7T, and strains M. zhoushanense KCTC 42782T and Marinobacterium lutimaris KACC 13703T, were grown on marine agar at 30 °C and 25 °C, respectively, for 48 h. The major fatty acids (>5% of the total fatty acids) are highlighted in bold. -, not detected; TR, trace amount (<1%). Data represent percentages of total fatty acids. a Data from Kang et al. [12].
Table
Table 3. Differential phenotypic features of strain D7T in comparison to those of the closest related species of the genus Marinobacterium.
Table 3. Differential phenotypic features of strain D7T in comparison to those of the closest related species of the genus Marinobacterium.
Characteristic12
Cell size (µm)0.5–0.6 × 1.0–1.80.4–0.6 × 1.0–2.0 a
Temperature range for growth (optimum) (°C)15–40 (37)15–43 (37–40) a
pH range for growth (optimum)5–9 (7)5.5–9.5 (6.5–7.5) a
NaCl range concentration for growth (optimum) (%, w/v)0–7.5 (3)0.25–9 (1–1.5) a
Nitrate reduction+-
Citrate utilization-+
Carbon source utilization in Biolog GEN III:
D-Cellobiose+-
Gentiobiose+-
D-Serine-+
D-Mannitol-+
D-Arabitol-+
D-Aspartic Acid+-
D-Glucuronic Acid-+
Mucic Acid-+
D-Saccharid Acid-+
Citric Acid+-
Sodium Butyrate+-
Enzymatic activities in API ZYM:
α-Glucosidase-+
Acid production in API 50CH:
D-Maltose+-
D-Sucrose-+
D-Trehalose-+
Strains: 1, Strain D7T; 2, Marinobacterium zhoushanense KCTC 42782T. All data from this study, except a that were obtained from Han et al. [15]. +, positive; -, negative. Other features included in the species description were common for both strains.
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Durán-Viseras, A.; Castro, D.J.; Reina, J.C.; Béjar, V.; Martínez-Checa, F. Taxogenomic and Metabolic Insights into Marinobacterium ramblicola sp. nov., a New Slightly Halophilic Bacterium Isolated from Rambla Salada, Murcia. Microorganisms 2021, 9, 1654. https://doi.org/10.3390/microorganisms9081654

AMA Style

Durán-Viseras A, Castro DJ, Reina JC, Béjar V, Martínez-Checa F. Taxogenomic and Metabolic Insights into Marinobacterium ramblicola sp. nov., a New Slightly Halophilic Bacterium Isolated from Rambla Salada, Murcia. Microorganisms. 2021; 9(8):1654. https://doi.org/10.3390/microorganisms9081654

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Durán-Viseras, Ana, David J. Castro, José Carlos Reina, Victoria Béjar, and Fernando Martínez-Checa. 2021. "Taxogenomic and Metabolic Insights into Marinobacterium ramblicola sp. nov., a New Slightly Halophilic Bacterium Isolated from Rambla Salada, Murcia" Microorganisms 9, no. 8: 1654. https://doi.org/10.3390/microorganisms9081654

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