Marinobacter azerbaijanicus sp. nov., a moderately halophilic bacterium from Urmia Lake, Iran

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published in International Journal of Systematic and Evolutionary Microbiology on 03 April 2024, available at: https://doi.org/10.1099/ijsem.0.006308 .” 1 Marinobacter azerbaijanicus sp. nov., a moderately halophilic bacterium from 1 Urmia Lake, Iran 2 3 4 Cristina Sanchez-Porro1†, Elnaz Mehdizadeh Aghdam2,3†, Soheila Montazersaheb2, Vahideh 5 Tarhriz4, Elham Kazemi3, Mohammad Ali Amoozegar5, Antonio Ventosa1*, Mohammad Saeid 6 Hejazi2,3* 7 8 1 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 9 Sevilla, Spain. 10 2 Molecular Medicine Research Center, Biomedicine Institute, Tabriz University of Medical 11 Sciences, Tabriz, Iran. 12 3 Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tabriz University of 13 Medical Sciences, Tabriz, Iran 14 4 Cardiovascular Center of Excellence, Louisiana State University Health Sciences Center, New 15 Orleans, Louisiana, USA 16 5 Extremophiles Laboratory, Department of Microbiology, School of Biology and Center of 17 Excellence in Phylogeny of Living Organisms, College of Science, University of Tehran, 18 Tehran, Iran. 19 20 * Corresponding authors: 21 Antonio Ventosa: [email protected] 22 Mohammad Saeid Hejazi: [email protected] 23 2 24 † These authors contributed equally to this study and should be considered as co-first authors. 25 26 Keywords: Marinobacter; Urmia Lake; halophilic bacteria; taxonomy; new taxa; comparative 27 genomics 28 29 Abbreviations: AAI, Average Amino acid identity; OrthoANI, Average Nucleotide Identity; 30 dDDH, digital DNA-DNA hybridization; GGDC, Genome-to-Genome Distance Calculator; 31 OGRI, Overall Genome Relatedness Indexes; DPG, diphosphatidylglycerol; PG, 32 phosphatidylglycerol; PE, phosphatidylethanolamine; GPL, glycophospholipid; Q-9, ubiquinone- 33 9. 34 The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and genome 35 sequence of strain TBZ242T are MH393200 and JASSVS000000000.1, respectively. 36 37 Abstract 38 A novel moderately halophilic, Gram-staining negative and facultatively anaerobic bacterium, 39 designated as strain TBZ242T, was isolated from water of Urmia Lake in Azerbaijan region of 40 Iran. The cells were found to be rod-shaped and motile by a single polar flagellum, producing 41 circular and yellowish colonies. The strain could grow in the presence of 0.5–10% (w/v) NaCl 42 (optimum, 2.5–5%). The temperature and pH ranges for growth were 15–45 ºC (optimum 30 ºC) 43 and pH 7.0–11.0 (optimum pH 8.0) on marine agar. The 16S rRNA gene sequence analysis 44 revealed strain TBZ242T belonged to the genus Marinobacter, showing the highest similarities to 45 Marinobacter algicola DG893T (98.8%), Marinobacter vulgaris F01T (98.8%), Marinobacter 46 3 salarius R9SW1T (98.5%), Marinobacter panjinensis PJ-16T (98.4%), Marinobacter orientalis 47 W62T (98.0%) and Marinobacter denitrificans JB2H27T (98.0%). The 16S rRNA and core- 48 genome phylogenetic trees showed that strain TBZ242T formed a distinct branch, closely related 49 to a subclade accommodating M. vulgaris, M. orientalis, M. panjinensis, M. denitrificans, M. 50 algicola, M. salarius and M. iranensis, within the genus Marinobacter. Average Nucleotide 51 Identity (OrthoANI) and Digital DNA-DNA hybridization (dDDH) percentages between strain 52 TBZ242T and the type strains of the related species of Marinobacter were ≤ 85.0% and 28.6%, 53 respectively, confirming that TBZ242T represents a distinct species. The major cellular fatty acids 54 of strain TBZ242T were C16:0 and C16:1 ω7C/C16:1 ω6C and the quinone was ubiquinone Q-9. The 55 genomic DNA G+C content of strain TBZ242T is 57.2 mol%. Based on phenotypic, 56 chemotaxonomic, and genomic analyses strain TBZ242T represents a novel species within the 57 genus Marinobacter, for which the name Marinobacter azerbaijanicus sp. nov. is proposed. The 58 type strain is TBZ242T (= CECT 30649T = IBRC-M 11466T). Genomic fragment recruitment 59 analysis showed that this species prefers aquatic saline environments with intermediate salinities, 60 being detected on metagenomic databases of Lake Meyghan (Iran) with 5% and 18% salinity, 61 respectively. 62 63 Introduction 64 Currently, the genus Marinobacter belongs to the family Marinobacteraceae [1] within the class 65 Gammaproteobacteria, phylum Pseudomonadota, and was first described by Gauthier et al. [2]. 66 This genus is composed of 60 species with validly published names 67 [https://lpsn.dsmz.de/genus/marinobacter; 3]. Most species of the genus Marinobacter are Gram- 68 stain-negative, rod-shaped, motile by flagella, and halophilic. Marinobacter members have been 69 4 isolated from various environments, such as marine sediments [4,5], saline soils [6], mangrove 70 sediments [7], marine salterns [8], surface seawater [9], or offshore sediments [10]. Members of 71 this genus require salt for their growth, thereby, they are slight or moderate halophiles [11-15]. 72 Many studies have pointed out the biodiversity and ecological significance of members of the 73 genus Marinobacter. In this regard, Anh et al. [16] showed the bioaugmentation ability of M. 74 nauticus (M. hydrocarbonoclasticus) for the removal of total nitrogen and chemical oxygen 75 demand from fish wastewater. Besides, the genus Marinobacter was found to be dominant among 76 the bacterial communities isolated from hydrocarbon-polluted regions for metabolizing 77 hydrocarbons [17]. In addition, most Marinobacter strains mediate the biosynthesis of wax esters, 78 which is mainly utilized in the industry [18]. Notably, some species have the capability to 79 metabolize inorganic elements such as iron, manganese, and arsenic [91] . These are examples of 80 the potential of members of Marinobacter to be used in biodegradation and biotechnological 81 processes. 82 Previous studies reported the diversity of halophilic bacteria in Urmia Lake [20-21]. This salty 83 lake is located in the uppermost northwest corner of Iran, between West- and East-Azerbaijan 84 (Azarbaijan) provinces. In our recent study [20], we isolated a novel cultivable, rod-shaped, motile, 85 pale yellow-pigmented colony producing bacterium, designated strain TBZ242T, that was 86 identified as a member of the genus Marinobacter. The present study aimed to determine the 87 phenotypic and genotypic characteristics to clarify the taxonomic position of strain TBZ242T 88 within the genus Marinobacter. Our data suggest that it constitutes a new species of this genus. 89 Besides, additional genomic and metagenomic studies permitted to determine some phenotypic 90 features and the ecological distribution of this new species. 91 92 5 Materials and methods 93 Isolation and cultivation 94 Strain TBZ242T was isolated from a water sample of Urmia Lake, located in Azerbaijan region, 95 between East and West Azerbaijan provinces at Northwest of Iran. The location of sampling was 96 near Sharafkhaneh (37°32′23.45′′N 45°26′25.11′′E). The salinity of the sample was 30.1% (w/v) 97 NaCl, the pH was 6.8 and the water temperature was 27 ºC [20]. For the isolation, 100 μl of the 98 brine sample was plated on marine agar (MA) (Becton-Dickinson) medium and incubated at 30 99 °C for about 3–4 days under aerobic conditions. The pH of the medium was adjusted to 7.5. Having 100 obtained the colony, the strain was cultured in marine broth (MB), and then plated on the same 101 agar medium in order to obtain a pure culture for further studies. The strain TBZ242T was stored 102 at –80 °C in MB medium containing 15% (v/v) glycerol. For comparative purposes, the following 103 strains were used as reference in this study: Marinobacter algicola DSM 16394T and Marinobacter 104 iranensis 71-iT. These strains were cultured under the same conditions than strain TBZ242T. 105 106 Phylogenetic and genomic analyses 107 At first step, the phylogenetic analysis of strain TBZ242T was conducted by comparison of the 16S 108 rRNA gene sequence. Having extracted the genomic DNA, the 16S rRNA gene was amplified by 109 polymerase chain reaction (PCR), using universal primers 27F (5'- 110 AGAGTTTGATCCTGGCTCAG-3') and 1492R (5' GGTTACCTTGTTACGACTT-3') [22]. The 111 PCR amplification was performed by 35 cycles of denaturation at 95 °C for 1 min, annealing at 55 112 °C for 75 sec and extension at 72 °C for 85 sec. The amplification procedure was preceded by 113 initial denaturation at 95 °C for 5 min and followed by final extension at 72 °C for 10 min [23]. 114 The PCR product was sequenced by Macrogen Inc., South Korea. The 16S rRNA gene sequence 115 6 identity of strain TBZ242T was analyzed and compared with the phylogenetic neighbours using 116 the EzBioCloud tool [24]. In addition, nBLAST toll (http://www.blast.ncbi.nlm.nih.gov) was used 117 for further similarity evaluation. Phylogenetic trees were constructed using three different 118 algorithms: maximum-parsimony [25], neighbour-joining [26] and maximum-likelihood [27], 119 integrated in the ARB software for phylogenetic inference [28]. The 16S rRNA gene sequences 120 from the type strains of the related species used for phylogenetic comparisons were obtained from 121 GenBank database. Bootstrap analysis (1000 replications) was performed to evaluate the 122 robustness of the phylogenetic trees [29]. 123 The genomic DNA of strain TBZ242T was extracted and purified by the method described by 124 Marmur [30] and was used to obtain its genome sequence by a whole-genome shotgun strategy with 125 Illumina NovaSeq 6000 platform and 150 bp paired-end sequencing reads (Novogene Europe, 126 Cambridge, United Kingdom). For de novo assembly of the reads Spades v.3.13.0. [31] was used. 127 The bioinformatic software CheckM v.1.0.5 [32] and Quast v.2.3 [33] were used to determine the 128 quality of the final contigs. The draft genome sequence of strain TBZ242T was deposited in 129 GenBank/EMBL/DDBJ under the accession number JASSVS000000000.1. For the phylogenomic 130 core genome analysis reconstruction all-versus-all BLAST search [34] was used for comparisons of 131 all predicted protein-coding genes annotated from each available genome. MUSCLE [35] was used 132 for the individual alignment of the core orthologous genes with diagonal optimization and adjusting 133 to the maximum number of iterations (default values for the other parameters). The phylogenomic 134 tree was reconstructed by using FastTree v.2.1 software [36]. Additionally, a phylogenomic tree was 135 constructed using a concatenated alignment of 120 bacterial conserved concatenated proteins 136 (Bac120sets) with GTDB-tk v.2.3.0 [37] by using FastTree v.2.1 [36]. Then, the phylogenetic tree 137 was modified using the online interactive Tree of Life (iTOL) [38]. 138 7 The genomic OGRI indexes Average Nucleotide Identity (OrthoANI) and digital DNA-DNA 139 hybridization (dDDH) between the genome of strain TBZ242T and the genomes of the related type 140 species of Marinobacter were calculated by using OAT software v.0.93.1 [39] and the Genome- 141 to-Genome Distance Calculator (GGDC) [40] website, using formula 2 [41], respectively. Finally, 142 the Average Amino acid Identity (AAI) index was determined using AAI CompareM program 143 [https://github.com/dparks1134/CompareM]. The genomic DNA G+C content of strain TBZ242T 144 and the other closely related strains were determined from their whole genome sequences. A 145 detailed functional annotation of the genome of strain TBZ242T was performed by the online tool 146 BlastKOALA [42], which assigned functional KEGG Orthology (KO) and KEGG pathways. 147 148 Phenotypic, physiological and biochemical characteristics 149 To determine the growth of strain TBZ242T in various media, it was cultivated in marine agar 150 (MA), nutrient agar (NA), Luria Bertani (LB), MGM [21] and R2A medium at 30 °C for 7 days. 151 The NaCl range and optimal growth was tested in marine broth medium without NaCl and with 152 0.5, 1.0 and 2.5-25% (w/v) at intervals of 2.5% (w/v) NaCl. In addition, the strain TBZ242T was 153 cultivated at different pH values (5.0–11.0 at intervals of 1 pH unit). The media pH values were 154 adjusted using 0.1 M citric acid/0.1 M sodium citrate buffer for pH 5.0; 0.1 M KH2PO4/0.1 M 155 NaOH for pH 6.0-8.0; 0.1 M NaHCO3/0.1 M Na2CO3 buffer for pH 9.0-10.0, and 0.05 M 156 Na2HPO4/0.1 M NaOH buffer for pH 11.0 [43]. The cell morphology and motility by flagella were 157 examined by transmission electron microscopy (TEM). A drop of the cell’s suspension was placed 158 on a carbon coated copper grid. After 5 minutes to allow the sample absorb to the grid it was 159 stained with 2% (w/v) uranyl acetate. After air drying, the sample was observed under TEM (Zeiss 160 LEO 906, 100 kV microscope). Motility was also observed under phase-contrast optical 161 8 microscopy. Gram-staining was carried out according to manufacturer’s instructions 162 (BioMerieux). Catalase and oxidase tests were carried out according to standard methods [44]. 163 Hydrolysis of gelatin, Tween 20, Tween 80, starch and esculin were determined according to 164 previously described methods [45]. Further biochemical and physiological characterization of 165 strain TBZ242T was conducted using API 20NE, API ZYM and API 50CH systems (BioMerieux). 166 Considering that the strain did not grow in the standard medium of API kits after 48 h incubation, 167 due to its halophilic nature, sterile NaCl was added to API media with a final concentration of 2% 168 (w/v). Then, the bacterial cells were suspended in this medium and used for analysis [46]. 169 170 Chemotaxonomic characteristics 171 Fatty acids composition was determined by mid-exponential growth phase cells cultivated on 172 Marine Agar supplemented with 3% (w/v) NaCl at 30 °C. The whole-cell fatty acids composition 173 of strain TBZ242T was determined by gas chromatography (Agilent 6850) with the MIDI 174 Microbial Identification System, version 6.1 [47, 48]. Polar lipids of strain TBZ242T were 175 analyzed at the Iranian Biological Resource Center (IBRC), using two-dimensional thin-layer 176 chromatography (TLC), based on the method described by Minnikin et al. [49]. The first and 177 second directions were developed using chloroform:methanol:water (65:25:4, v/v/v) and 178 chloroform:methanol:acetic acid:water (80:12:15:4, v/v/v/v) solvents, respectively. The detection 179 of the polar lipids was carried out using the following reagents: molybdophosphoric acid, specific 180 for total polar lipids, ninhydrin, specific for aminolipids, molybdenum blue reagent, specific for 181 phospholipids, Dragendorff for quaternary nitrogen, and α-naphthol/sulfuric acid, specific for 182 glycolipids. Four TLC plates were prepared for each sample. The respiratory quinone content for 183 strain TBZ242T was analyzed at the Iranian Biological Resource Center, according to the method 184 15 Cells are Gram-stain-negative, 0.5 x 2-8 µm, rod-shaped, and motile by a polar flagellum. Colonies 323 were circular, convex and yellowish-pigmented. Facultatively anaerobic. Growth occurs at 15–45 324 ºC (optimum at 30 ºC). The range of pH was between 7.0–11.0 (optimum, pH 8.0), and in the 325 presence of 0.5–10% (w/v) NaCl after 48 hours and was observed up to 15% after one week. 326 Optimum growth occurred between 2.5-5% (w/v) NaCl in marine medium. Catalase and oxidase 327 positive. Nitrate was reduced to nitrite. Starch, esculin and Tweens 20 and 80 were hydrolyzed, 328 but not gelatin. Urease was negative. Positive for assimilation of glucose, mannitol, potassium 329 gluconate, adipic acid, malate, trisodium citrate, phenyl acetic acid and maltose (weak). Alkaline 330 phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine 331 arylamidase, cystine arylamidase, acid phosphatase (weak), naphthol-AS-BI-phosphohydrolase, 332 α-glucosidase and N-acetyl-ß-glucosaminidase activities were positive. The major cellular fatty 333 acids were C16:0 and C16:1 ω7C/C16:1 ω6C. The polar lipids included diphosphatidylglycerol (DPG), 334 phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and glycophospholipid (GPL). The 335 only respiratory quinone was ubiquinone-9 (Q-9). 336 The type strain is TBZ242T (= CECT 30649T = IBRC-M 11466T), isolated from a brine sample of 337 Urmia Lake, Iran. The type strain has a G+C content of 57.2 mol% in the genomic DNA. The 338 GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and the whole genome 339 sequences of strain TBZ242T are MH393200 and JASSVS000000000.1, respectively. 340 341 Funding information 342 This work was financially supported by the Molecular Medicine Research Center, Tabriz 343 University of Medical Sciences, Tabriz, Iran, [Pazhoohan ID: 68140 and Code: 344 IR.TBZMED.VCR.REC.1400.253], grant PID2020-118136GB-I00, funded by 345 16 MCIN/AEI/10.13039/501100011033, and grants P20_01066 and BIO-213, funded by the Junta de 346 Andalucía, both with FEDER funds. 347 348 Conflicts of interest 349 The authors declare that there are no conflicts of interest. 350 351 References 352 1. Liao H, Lin X, Li Y, Qu M, Tian Y. Reclassification of the taxonomic framework of 353 orders Cellvibrionales, Oceanospirillales, Pseudomonadales, and Alteromonadales in 354 class Gammaproteobacteria through phylogenomic tree analysis. mSystems 355 2020;5:e00543-20. 356 2. 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Metagenomic 511 sequence of prokaryotic microbiota from an intermediate-salinity pond of a saltern in Isla 512 Cristina, Spain. Genome Announc 2014;2:e00045-14. 513 64. Ghai R, Pašić L, Fernández AB, Martin-Cuadrado A-B, Mizuno CM, et al. New 514 abundant microbial groups in aquatic hypersaline environments. Sci Rep 2011;1:135. 515 65. Ng HJ, López-Pérez M, Webb HK, Gomez D, Tomoo Sawabe T, et al. Marinobacter 516 salarius sp. nov. and Marinobacter similis sp. nov., isolated from sea water. PLoS One 517 2014:9:e106514. 518 519 520 24 Legends to Figures 521 522 Figure 1. Maximum likelihood phylogenetic tree based on 16S rRNA gene sequences showing 523 the relationships between strain TBZ242T and its related taxa. Sequence accession numbers are 524 shown in parentheses. Bootstrap percentages are based on 1000 pseudoreplicates and only those 525 ≥70 % are shown at branch points. The species Halospina denitrificans HGD 1-3T was used as an 526 outgroup. Bar represents expected substitutions per nucleotide position. 527 528 Figure 2. Approximately maximum-likelihood phylogenomic tree based on the comparison of 529 1269 core-orthologous proteins showing the relationship between strain TBZ242T, related species 530 of the genus Marinobacter and three MAGs closely related to strain TBZ242T. Sequence accession 531 numbers are shown in parentheses. Branch support percentages are computed with the Shimodaira- 532 Hasegawa test and are shown at branch points. Halospina denitrificans DSM 15505T was used as 533 an outgroup. Bar, 0.05 substitutions per amino acid position. 534 535 Figure 3. Heatmap displaying Orthologous Average Nucleotide Identity (OrthoANI) and digital 536 DNA-DNA hybridization (dDDH) percentages among strain TBZ242T and the type strains of 537 species of the genus Marinobacter. 538 539 Figure 4. Heatmap showing the Average Amino acid Identity (AAI) percentages among strain 540 TBZ242T and the type strains of species of the genus Marinobacter. 541 542 31 Figure S4. Heatmap displaying Orthologous Average Nucleotide Identity (OrthoANI) (upper right) and digital DNA-DNA hybridization (dDDH) (lower left) percentages among Marinobacter azebaijanicus TBZ242T and the closest related strains/sequences of Marinobacter determined by GTDB-tk. 32 Figure S5. Heatmap showing the Average Amino acid Identity (AAI) percentages among strain Marinobacter azebaijanicus TBZ242T and the closest related strains/sequences of Marinobacter determined by GTDB-tk. 33 Figure S6. Electron photomicrograph of strain TBZ242T, showing the polar flagellum. Bar, 1 µm.