Structure of the 4-O-[1-Carboxyethyl]-d-Mannose-Containing O-Specific Polysaccharide of a Halophilic Bacterium Salinivibrio sp. EG9S8QL

The moderately halophilic strain Salinivibrio sp. EG9S8QL was isolated among 11 halophilic strains from saline mud (Emisal Salt Company, Lake Qarun, Fayoum, Egypt). The lipopolysaccharide was extracted from dried cells of Salinivibrio sp. EG9S8QL by the phenol–water procedure. The OPS was obtained by mild acid hydrolysis of the lipopolysaccharide and was studied by sugar analysis along with 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, and HMBC experiments. The OPS was found to be composed of linear tetrasaccharide repeating units of the following structure: →2)-β-Manp4Lac-(1→3)-α-ManpNAc-(1→3)-β-Rhap-(1→4)-α-GlcpNAc-(1→, where Manp4Lac is 4-O-[1-carboxyethyl]mannose.


Introduction
Microorganisms adapted to extreme environmental conditions, unsuitable for the survival of other organisms, are called extremophiles. They include archaea, halophilic and halotolerant bacteria, and algae. Among other factors, salinity strongly affects the composition of the microbial communities of water areas [1,2]. Saline environments are found everywhere on Earth and include seas, thermal springs, salterns, and lakes. In endorheic lakes, which are not connected to the world ocean by river systems, water is lost through evaporation or percolation. As a result, minerals and other erosion products accumulate. Endorheic lakes are used widely to produce mineral salts. The unique hydrological regime, seasonal changes in salinity, and human activities greatly affect microbial diversity in such lakes [3,4].
Moderately halophilic Gram-negative gammaproteobacteria of the genus Salinivibrio within the family Vibrionaceae are normal inhabitants of hypersaline systems including marine environments worldwide [5,6]. They have been isolated from saline lakes, soils,

LPS Isolation and Characterization
The LPS was isolated from the cells Salinivibrio sp. EG9S8QL by hot phenol-water extraction [22]. The fatty acid composition of the LPS obtained by GC analysis of their methyl ester derivatives showed the prevalence of 3-hydroxydodecanoic acid [ [20]. SDS-PAGE of the LPS demonstrated the presence of molecules containing the OPS, as well as intensively stained fast-migrating fractions corresponding to the R-form (Supplementary Material, Figure S2). The electrophoretic profile of the LPS was typical for molecules with relatively short O-chains.

Structural Analysis of the OPS
The LPS of Salinivibrio sp. EG9S8QL was obtained from dried cells by the phenolwater extraction and was degraded under mild acidic conditions. After the removal of lipid A by centrifugation, the OPS-containing supernatant was fractionated by gel-permeationchromatography on a Sephadex G-50 F column. The yield of the OPS fraction was 29% of the LPS mass. The GLC sugar analysis of alditol acetates obtained after hydrolysis of the OPS in 2 M TFA showed the presence of rhamnose (Rha), mannosamine (ManN) and glucosamine (GlcN) as the major components, in a peak area ratio of 1.0:0.5:0.6. Additionally, the GC-MS analysis of the acetylated methyl glycosides showed the presence of a hexose carrying 2-hydroxypropanoyl residue, identified later by NMR as 4-O-[1-carboxyethyl]-D-mannose (see below). The electron impact EI mass spectrum of the latter compound in a lactone form as an acetylated methyl glycoside showed characteristic ions at m/z 301, 272, 259, 230, and 142 ( Figure 1). GC of the acetylated (S)-2-octyl glycosides revealed the L absolute configuration of Rha and the D configuration of other monosaccharides.
Deacylation of the LPS with NH4OH revealed that 12:0(3-OH) is a primary O-linked fatty acid. Taking into account the conservativeness of the lipid A structure within each bacterial genus, we can suggest that the lipid A of Salinivibrio sp. EG9S8QL is structurally close to that of S. sharmensis BAG T [20]. SDS-PAGE of the LPS demonstrated the presence of molecules containing the OPS, as well as intensively stained fast-migrating fractions corresponding to the R-form (Supplementary Material, Figure S2). The electrophoretic profile of the LPS was typical for molecules with relatively short O-chains.

Structural Analysis of the OPS
The LPS of Salinivibrio sp. EG9S8QL was obtained from dried cells by the phenolwater extraction and was degraded under mild acidic conditions. After the removal of lipid A by centrifugation, the OPS-containing supernatant was fractionated by gelpermeation-chromatography on a Sephadex G-50 F column. The yield of the OPS fraction was 29% of the LPS mass. The GLC sugar analysis of alditol acetates obtained after hydrolysis of the OPS in 2 M TFA showed the presence of rhamnose (Rha), mannosamine (ManN) and glucosamine (GlcN) as the major components, in a peak area ratio of   The structure of the OPS was examined by NMR spectroscopy. The major series of 1 H NMR spectrum of the OPS (Figure 2) contained signals for four anomeric protons at δ 4.82-5.07, two CH 3 -C groups (H-6 of Rha) at δ 1.33 and H-3 of lactic acid (Lac) at δ 1.40, signals of N-acetyl groups at δ 2.07, and other sugar protons at δ 3.45-4.60. Along with the major series in the 1 H NMR spectrum, a minor series of signals was observed, which presumably originated from the core oligosaccharide attached to the O-chain.  Table 1.
The 1 H and 13 C NMR spectra of the OPS were assigned using 1 H, 1 H COSY, TOCSY, ROESY, 1 H, 13 C HSQC (Figure 4), and HMBC experiments (Table 1). Based on 13 C NMR chemical shifts, intraresidue 1 H, 1 H and 1 H, 13  and H-2/H-3,4,5,6 cross-peaks for units A-C and H-1/H-2,3,4,5,6 cross-peaks for unit D. The signals within each spin system were assigned using the 1 H, 1 H-COSY spectrum. chemical shifts, intraresidue 1 H, 1 H and 1 H, 13 C correlations and 3 JH,H coupling constant values, spin systems of Lac and four monosaccharide residues were identified, including three manno-configurated sugars, D-Manp4Lac, D-ManpNAc and L-Rhap (units A, B and C), and one gluco-configurated sugar, D-GlcpNAc (unit D). The TOCSY spectrum showed H-1/H-2 and H-2/H-3,4,5,6 cross-peaks for units A-C and H-1/H-2,3,4,5,6 cross-peaks for unit D. The signals within each spin system were assigned using the 1 H, 1 H-COSY spectrum.  Table 1.     Table 1. The presence of Lac at position 4 of residue A was confirmed on the basis of a strong correlations between A H-4 and C-2 of Lac at δ 3.60/79.8 and vice versa, H-2 of Lac and A C-4 at δ 4.00/78.3 in the 1 H, 13 C HMBC spectrum.
The α configuration of units B and D and the β configuration of units A and C were inferred from characteristic chemical shifts of the C-5 signals as compared with published data of the corresponding αand β-methyl glycosides [23].
The linkage analysis of sugar residues in the OPS was performed using 1 H, 1 H-ROESY and 1 H, 13 [23,24], confirmed the glycosylation pattern and revealed the monosaccharide sequence in the repeating unit.
Mar. Drugs 2021, 19, x The presence of Lac at position 4 of residue A was confirmed on the basis of correlations between A H-4 and C-2 of Lac at δ 3.60/79.8 and vice versa, H-2 of L C-4 at δ 4.00/78.3 in the 1 H, 13 C HMBC spectrum.
The α configuration of units B and D and the β configuration of units A and inferred from characteristic chemical shifts of the C-5 signals as compared with p data of the corresponding α-and β-methyl glycosides [23].
The linkage analysis of sugar residues in the OPS was performed using 1 H, 1 H and 1 H, 13 13 C HMBC spectrum of the OPS from Salinivibrio sp. EG9S8QL. Arabic numerals before and after slash refer to protons and carbons, respectively, in sugar residues denoted by letters as indicated in Table 1

Discussion
Living in the extreme conditions of saline lakes, which are characterized by a higher salt concentration than seawater, a high level of ultraviolet radiation and heavy metals, has led to a diversification of adaptive mechanisms in halophilic microorganisms. These include production of polysaccharides and other biopolymers and/or modification of their structure. A high level of extracellular polysaccharide production increases water retention and heavy metals binding [16]. The ratio between proteins and polysaccharides of the bacterial surface and their structural features is essential for physicochemical characteristics of cell surface such as hydrophobicity, surface charge, etc., important for the adhesion of bacteria to various surfaces, intercellular aggregation and biofilm  13 C HMBC spectrum of the OPS from Salinivibrio sp. EG9S8QL. Arabic numerals before and after slash refer to protons and carbons, respectively, in sugar residues denoted by letters as indicated in Table 1 13 C HMBC spectrum of the OPS from Salinivibrio sp. EG9S8QL. Arabic numerals before and after slash refer to protons and carbons, respectively, in sugar residues denoted by letters as indicated in Table 1.
Therefore, the OPS consists of linear tetrasaccharide repeating units (Figure 7):

Discussion
Living in the extreme conditions of saline lakes, which are characterized by a higher salt concentration than seawater, a high level of ultraviolet radiation and heavy metals, has led to a diversification of adaptive mechanisms in halophilic microorganisms. These include production of polysaccharides and other biopolymers and/or modification of their structure. A high level of extracellular polysaccharide production increases water retention and heavy metals binding [16]. The ratio between proteins and polysaccharides of the bacterial surface and their structural features is essential for physicochemical characteristics of cell surface such as hydrophobicity, surface charge, etc., important for the adhesion of bacteria to various surfaces, intercellular aggregation and biofilm

Discussion
Living in the extreme conditions of saline lakes, which are characterized by a higher salt concentration than seawater, a high level of ultraviolet radiation and heavy metals, has led to a diversification of adaptive mechanisms in halophilic microorganisms. These include production of polysaccharides and other biopolymers and/or modification of their structure. A high level of extracellular polysaccharide production increases water retention and heavy metals binding [16]. The ratio between proteins and polysaccharides of the bacterial surface and their structural features is essential for physicochemical characteristics of cell surface such as hydrophobicity, surface charge, etc., important for the adhesion of bacteria to various surfaces, intercellular aggregation and biofilm formation [25,26]. The predominant component of the outer membrane of Gram-negative bacteria, lipopolysaccharide, makes a significant contribution to the formation of a negative charge on the bacterial cell surface, facilitating the electrostatic binding of polyvalent cations and promoting cell aggregation [27].
Glycans of extremophiles are often unique in structure and properties [28] but their structural and functional roles, including in the process of haloadaptation, have not been studied enough. Studies of the membrane and extracellular polysaccharide structures and physicochemical properties can make it possible to advance the understanding of both the role of these glycans in the adaptation of microorganisms to extreme conditions for existence and predict the possibility of their use for biotechnological purposes.
In this study, we determined the structure of the OPS from the moderately halophilic Gram-negative bacterium Salinivibrio sp. 9S8 isolated from Lake Qarun. The OPS has a linear regulate structure and is composed of tetrasaccharide repeating units. To our knowledge, this structure has not previously been found in bacterial polysaccharides [21]. A similar tetrasaccharide repeated unit decorated with pyruvate at 4,6 positions of Man was found in the OPS of Raoultella terrigena and Shigella dysenteriae Type 10 [29,30] as well as in the CPS from Escherichia coli O8:K50:H [31]. D-Manp4Lac is a rather rare component of bacterial polysaccharides. Until now, it has not been found in the OPS, but was reported as a component of the extracellular polysaccharides from Cyanospira capsulate, Mycobacterium lacticolum121 and Mycobacterium album B-88 [32][33][34].
Non-carbohydrate organic acids are often found in bacterial lipopolysachharides, for example, representatives of the Proteus, Providencia, Shigella [35,36]. It is believed that the presence of non-carbohydrate substituents and branching points in the polysaccharide chain increase the immunogenicity of the O antigen.

Sampling and Strain Isolation
The strain Salinivibrio sp. EG9S8QL was isolated from a saline mud sample (salt mixed with clay collected from the bottom of solar salt ponds), collected in August 2018 from the Emisal salt company, Lake Qarun, Fayoum, Egypt (29 • 28 19.92" N and 30 • 36 51.12" E). Salt concentration of the tested sample was 18%. Strain EG9S8QL was isolated using S-G medium [37] supplemented with 1% glucose. The inoculated flask was incubated at 35 • C for 2 days at 200 rpm on a rotary shaker. Then, 0.1 mL of serial dilutions from the enriched sample was spread onto the surface of S-G agar plate medium and then incubated in sealed plastic bags at 35 • C for 2-3 days. Eleven isolates were isolated from the enriched sample. Strain EG9S8QL was selected for further phenotypic and genotypic characterizations. The growth factors (pH, NaCl content, temperature, carbon sources, cultivation duration) were studied to obtain the maximum bacterial growth as described [38]. The culture was stored at −80 • C in an S-G medium with final concentration 20% of glycerol.

Phenotypic and Genotypic Characterization of Strain EG9S8QL
The colony morphology of isolate EG9S8QL was described. Gram staining and the KOH string [39] tests were conducted. Bacterial motility and cell morphology were examined using phase-contrast microscopy with a DM2500 instrument (Leica, Germany). Hydrolysis of casein, gelatin and starch were determined as described [40]. Nitrate and nitrite reduction were tested as recommended [41]. Catalase activity was determined as described [42]. Other enzymatic activities were tested using API-20E (bioMérieux SA, Lyon, France) and BIOLOG GEN III MicroPlate TM systems (BIOLOG TM , Cabot Blvd., Hayward, Berkeley Heights, NJ, USA), according to the instructions with 5% (w/v) NaCl.
Strain EG9S8QL was identified by analyzing the sequence of the gene encoding 16S rRNA (16SrDNA). Genomic DNA extraction, amplification of 16S rRNA gene using universal primers fD1 (AGAGTTTGATCCTGGCTCAG) and rD1 (AAGGAGGTGATCCAGCC) [43], and purification of PCR product were conducted as described [38]. The nucleotide sequence of the purified PCR product was determined using an ABI 3730xL automated Mar. Drugs 2021, 19, 508 9 of 12 DNA sequencer (Applied Biosystems, Waltham, MA, USA) through Lab Biotechnology Company (Cairo, Egypt). The 16S rRNA gene sequences were initially analyzed using the program BLAST (National Center Biotechnology Information, http://www.ncbi.nml. nih.gov, 27 December 2018). The phylogenetic tree was designed as described [44] using Escherichia coli (NR114042.1) as an outgroup. Data of 16S rRNA gene sequence of strain EG9S8QL have been deposited in GenBank under accession number MZ646069.

Isolation of the Lipopolysaccharide and O-Specific Polysaccharide
The bacteria were cultivated in GRM medium supplemented with 5% NaCl to late exponential phase, and cells were harvested by centrifugation. The cells were repeatedly washed with 0.15 M NaCl, desiccated with acetone and air-dried. The biomass (8.8 g) was extracted by the Westphal procedure [21], proteins and nucleic acids were precipitated by CCl 3 CO 2 H (pH 2.7) and removed by centrifugation. After dialysis of the supernatant, an LPS preparation was obtained in 2.65% yield. The LPS was subjected to electrophoresis in 13% SDS polyacrylamide gel [45]. The components were visualized by staining the gels with a silver nitrate based dye [46]. An LPS sample (65 mg) was hydrolyzed with aq 2% AcOH at 100 • C for 3 h, a lipid precipitate was removed by centrifugation (13,000× g, 20 min), and the carbohydrate portion was fractionated by GPC on a column (56 × 2.6 cm) of Sephadex G-50 Superfine in 0.05 M pyridinium acetate buffer pH 4.5. The elution was monitored by a Knauer differential refractometer (Berlin, Germany). A high-molecular-mass OPS preparation was obtained in 29.2% yield.

Monosaccharide Analyses
The monosaccharides were analyzed as the alditol acetates after hydrolysis of the OPS with 2 M CF 3 CO 2 H (120 • C, 2 h) [47] and acetylated methyl glycosides after methanolysis of the OPS with acetylchloride in methanol (1:10, v/v, 100 • C, 4 h) by GC on an HP-5ms capillary column using an Agilent 7820A GC system and a temperature gradient of 160 • C (1 min) to 290 • C at 7 • C min −1 . The absolute configurations of the monosaccharides were determined by GC of the acetylated glycosides with (S)-2-octanol, as described [48]. GC-MS was performed on a Hewlett Packard 5890 chromatograph (Palo Alto, CA, USA) equipped with a HP-5MS column and connected to a Hewlett Packard 5973 mass spectrometer (Palo Alto, CA, USA).

NMR Spectroscopy
1 H and 13 C NMR spectra were recorded on a Bruker Avance-III spectrometer (700.13 MHz for 1 H and 176.04 MHz for 13 C) using a 5 mm broadband inverse probehead for solutions in 99.95% D 2 O after deuterium exchange by freeze-drying sample solutions in 99.9% D 2 O. Acetone (δ C 31.45, δ H 2.225) was used as the internal calibration standard. The 2D NMR spectra were obtained using standard Bruker software, and Bruker TOPSPIN 2.1 program (Bruker Corporation, Billerica, MA, USA) was used to acquire and process the NMR data. The 2D TOCSY and ROESY spectra were recorded with a 180 ms duration of MLEV-17 spin-lock and a 200 ms mixing time, respectively. 1 H, 13 C HMBC was optimized for an 8 Hz long-range constant.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/md19090508/s1, Figure S1: Neighbour-joining tree showing the phylogenetic position of strain EG9S8QL (with blue color) and its related neighbour strains base d on 16S rRNA gene sequences. Bootstrap values (expressed as percentages of 100 replications) are shown at branch points. Bar 0.1 sub-stitutions per nucleotide position, Figure S2: Silver-stained SDS PAGE of the LPS from Salinivibrio sp. EG9S8QL (20 µg), Table S1: Comparative analysis of phenotypic features of strain EG9S8QL and closely related Salinivibrio species.