1 Pseudomonas khazarica sp. nov., a polycyclic aromatic hydrocarbon- 2 degrading bacterium isolated from Khazar Sea sediments 3 4 Vahideh Tarhriz1†, Imen Nouioui2†, Cathrin Spröer3, Susanne Verbarg3, Vida Ebrahimi4, 5 Carlos Cortés-Albayay2, Peter Schumann3, Mohammad Amin Hejazi5, Hans-Peter Klenk2*, 6 Mohammad Saeid Hejazi1,4,6* 7 8 9 1 Molecular Medicine Research Center, Biomedicine Institute, Tabriz University of Medical 10 Sciences, Tabriz, Iran 11 2 School of Natural and Environmental Sciences, Newcastle University, Newcastle upon 12 Tyne, UK 13 3 Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, 14 Inhoffenstr. 7B, 38124 Braunschweig, Germany 15 4 Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran 16 5 Branch for the Northwest and West Region, Agriculture Biotechnology Research Institute of 17 Iran (ABRII), Tabriz, Iran 18 6 School of Advanced Biomedical Sciences, Tabriz University of Medical Sciences, Tabriz, 19 Iran 20 21 *Authors for correspondence 22 Hans-Peter Klenk 23 School of Natural and Environmental Sciences 24 Newcastle University 25 Ridley Building 2 26 Newcastle upon Tyne 27 NE1 7RU 28 UK 29 Telephone: +44 (0) 191 208 5138 30 Fax: 31 Email: [email protected] 32 33 Mohammad Saeid Hejazi 34 Molecular Medicine Research Center 35 Tabriz University of Medical Sciences 36 Tabriz, 37 Iran 38 Tel: +98 (411) 3372256 39 Fax: +98 (411) 334 4798 40 Email: [email protected] 41 †: These authors contributed equally in this study and should be considered as co-first 42 authors. 43 1 44 Abstract 45 A novel Gram-negative, aerobic, motile and rod-shaped bacterium with the potential to 46 biodegrade polycyclic aromatic hydrocarbons, was isolated from Khazar (Caspian) Sea. 47 Strain TBZ2T grows in the absence of NaCl and tolerates up to 8.5% NaCl and could grow at 48 pH 3.0-10.0 (optimum, pH 6.0-7.0) at 10- 45°C (optimum, 30 °C). The major fatty acids are 49 C18:1 ω7C, C16:1 ω7C/ 15 iso OH, C16:0, C12:0 3-OH, C10:0 3-OH. 16S rRNA gene sequence 50 analysis showed that strain TBZ2T is a member of the genus Pseudomonas with the highest 51 similarity to P. oleovorans subsp. oleovorans DSM 1045T (98.83%), P. mendocina NBRC 52 14162T (98.63%), P. oleovorans subsp. lubricantis RS1T (98.61%) and P. alcaliphila JCM 53 10630T (98.49%) based on EzBioCloud server. Phylogenetic analyses using housekeeping 54 genes (16S rRNA, rpoD, gyrB and rpoB) and genome sequences demonstrated that the strain 55 TBZ2T formed a distinct branch closely related to the type strains of P. mendocina and P. 56 guguanensis. Digital DNA-DNA hybridization and average nucleotide identity values 57 between strain TBZ2T and its closest relatives, P. mendocina NBRC 14162T (25.3%, 81.5%) 58 and P. guguanensis ICM 18146T (26.8%, 79.0%), rate well below the designed threshold for 59 assigning prokaryotic strains to the same species. On the basis of phenotypic, 60 chemotaxonomic, genomic and phylogenetic results, it is recommended that strain TBZ2T is a 61 novel species of the genus Pseudomonas, for which the name Pseudomonas khazarica sp. 62 nov., is proposed. The type strain is TBZ2T (=LMG 29674T =KCTC 52410T). 63 Keywords: Pseudomonas khazarica, polycyclic aromatic hydrocarbons, Khazar 64 65 66 67 68 2 69 Introduction 70 Polycyclic Aromatic Hydrocabons (PAHs), a group of environmental organic pollutants 71 with crucial public health concern due to their genotoxic and carcinogenic properties, 72 unfortunately are found in high concentrations in many industrial sites (Martins et al. 2016; 73 Wilson and Jones 1993). They are considered as environmental concerns due to their stability 74 against degradation and their potential to bioaccumulate in the food chain (Bosma et al. 1996; 75 Fujikawa et al. 1993). Various bacteria, isolated from marine habitats, have been reported to 76 remediate and degrade PAHs, including naphthalene, anthracene and phenanthrene (Ghosal et 77 al. 2016; Jin et al. 2015). During an analysis of the microbial biodegradation ability of 78 polycyclic aromatic hydrocarbons (PAHs) in Khazar (Caspian) Sea, the world's largest lake, a T 79 Gram-negative, rod shaped and motile bacterium was isolated. The isolated strain, TBZ2 P P 80 (abbreviation of Tabriz) was subjected to a polyphasic taxonomic analysis. Based on the 81 results, this strain is considered to represent a novel species of the genus Pseudomonas. 82 The genus Pseudomonas, belonging to the family Pseudomonadaceae, was first proposed 83 by Migula (1894). Numerous members of the genus have been isolated from different 84 environmental sources such as soil, plants, animals and water and some of them are human 85 and plant pathogens (Busquets et al. 2017; Palleroni 1984). The nutritional spectrum of 86 aerobic Pseudomonas revealed that the ability of members of this genus in using different 87 compounds as the sole sources of carbon and energy has provided an essential platform for 88 phenotypic characterization (Palleroni 1984; Stanier et al. 1966). Based on 16S rRNA 89 phylogenetic study, P. oleovorans subsp. oleovorans is the closest related species to our 90 isolate with 98.79% similarity. P. oleovorans was isolated first time from water-oil 91 emulsions used as lubricants and cooling agents. It is a Gram-negative and methylotrophic 92 bacterium with the ability to use one-carbon compounds, such as methanol (Waturangi et al. 93 2011). 3 94 Materials and Methods 95 Isolation and preservation 96 Strain TBZ2T was isolated from estuary of Shahrood river (Qaemshahr area, 97 Mazandaran province, Iran) flowing north through the Alborz mountain into the Khazar 98 (Caspian) Sea, on ONR7a medium supplemented with one of PAHs compounds (including 99 naphthalene or phenanthrene or anthracene) as the carbon source (Tarhriz et al. 2014). 100 Growth was studied on brain heart infusion Agar (Scharlau) and MacConkey agar (Merck) 101 also on sea water agar, nutrient agar, LB (Luria-Bertani) media. Gram reaction was tested 102 (Gerhardt 1994) and confirmed by KOH lysis test (Gregersen 1978). 103 104 Phenotypic characterization 105 Growth was observed at various NaCl concentrations in lab made marine agar medium 106 containing (per liter): peptone, 5.0 g; yeast extract, 1.0 g; Fe3+-citrate, 0.1 g; NaCl, 19.45 g; 107 MgCl2 (dried), 8.8 g; Na2SO4, 3.24g; CaCl2, 1.8 g; KCl ,0.55 g; NaHCO3, 0.16 g; KBr, 0.08 108 g; SrCl2, 34.0 mg; H3BO3, 22.0 mg; Na-silicate, 4.0 mg; NaF, 2.4 mg; (NH4)NO3, 1.6 mg; 109 Na2HPO4, 8.0 mg; agar 15.0 g and pH was adjusted to 7.6 ± 0.2, supplemented without NaCl 110 and with 0.5-10% (w/v) NaCl at intervals of 0.5%. 111 Growth was investigated at 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 °C on lab made marine 112 broth. In order to determine the optimal growth temperature, samples were measured by UV 113 absorbance at 600 nanometer wave length (OD: 600) after 48 hours. The pH tolerance was 114 determined in marine broth with at 30°C (optimum condition) with the pH set from 4.0 and 115 12.0 (using increments of 1 pH unit). Also the cells were incubated in an anaerobic chamber 116 for 48 hours in order to determine the extent of growth under anaerobic conditions. 117 Transmission electron microscopy (TEM; Zeiss LEO906) imaging was conducted at an 4 118 acceleration voltage of 100 kV from 200 µl of a suspension of the bacterium at logarithmic 119 growth phase in 3 ml of distilled water. 120 Biochemical tests containing determination of oxidase and catalase activity, gelatin 121 liquefaction, ability to hydrolyze starch, Tween 20, Tween 80 and casein, production of 122 indole and H2S, nitrate reduction and motility were done as described by MacFaddin (2000). 123 The ability of strain TBZ2T in utilizing various carbon sources, the production of acid from 124 sugars and its physiological profile were tested using the API 20E, API 20NE and API 50CH 125 kits (bioMérieux Corporate). 126 Extraction and analysis of fatty acids were carried out using minor modifications of the 127 method of (Miller 1982) and (Kuykendall et al. 1988). The fatty acid methyl esters mixtures 128 were separated using Sherlock Microbial Identification System (MIS) (MIDI, Microbial ID, 129 Newark, DE 19711 U.S.A.) which followed by naming and percentages calculation by using 130 MIS Standard Software (Microbial ID). 131 132 Molecular characterization 133 For genotypic characterization, DNA was extracted from the strain TBZ2T by using the 134 method described by Corbin and colleagues (Corbin et al. 2001) with some modifications. 135 For phylogenetic analysis based on 16S rRNA gene sequence, 16S rDNA was targeted for 136 amplification by polymerase chain reaction (PCR) in the presence of forward 16F 5’-AGA 137 GTT TGA TCC TGG CTC AG- 3' and reverse 16R for Gram-negative bacteria; 5' -ACG 138 GCT ACC TTG TTA CGA CTT- 3' (Karlson et al. 1993; Tarhriz et al. (2011)) and was 139 sequenced by Bioneer Company (Korea). BLAST of the 16S rRNA gene sequence of strain 140 TBZ2T in the EzBioCloud’s identity service (Yoon et al. 2017a) lead to retrieve its closest 141 phylogenetic neighbours. Maximum-likelihood (ML) (Kimura 1980) and Maximum- 142 parsimony (MP) (Fitch 1971) phylogenetic trees, as well as the pairwise sequence similarities 5 143 between TBZ2T and its relatives, were performed using the DSMZ phylogenomics pipeline 144 available at Genome-to-Genome distance calculator (GGDC) web server 145 (http://ggdc.dsmz.de). 146 Multilocus sequence analyses (MLSA) were carried out based on concatenated partial 147 sequences of rpoD (RNA polymerase subunit D), gyrB (DNA gyrase subunit B), and rpoB 148 (RNA polymerase beta subunit) and 16S rRNA genes.