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. All the gene sequences cited above of
149 strain TBZ2T were obtained from the draft genome sequence (accession number
150 SBHZ00000000) while those of the nearest neighbours were retrieved from the GenBank
151 database. The type species of the genus Acinetobacter, Acinetobacter calcoaceticus
152 (Beijerinck 1911) was included as an outgroup in all the analyses. Multiple sequence
153 alignment program CLUSTALW (Thompson et al. 1994) in MEGA 7 (Molecular
154 Evolutionary Genetics Analysis version 7) software were used. ML and MP MLSA-based
155 phylogenetic trees were constructed using the same pipeline cited above. The genetic
156 distances between the loci of strain TBZ2T and those of its relatives were estimated using
157 Kimura 2-parameter (Kimura 1980).
158 A phylogenomic tree was constructed using the Type Strain Genome Server (TYGS),
159 available under https://tygs.dsmz.de, for a whole genome-based taxonomic analysis (Meier-
160 Kolthoff and Göker 2019).
161 Genome sequence analyses
162 The genomic DNA extraction, purification, quantification and DNA libraries were prepared
163 following the standard pipeline of MicrobesNG service (https://microbesng.uk/microbesng-
164 faq/). Genome sequencing was performed using Illumina HiSeq with 250 bp paired end
165 protocol. The Reads were trimmed using Trimmomatic 0.30 with a sliding window quality
166 cutoff of Q15 (Bolger et al. 2014). De novo assembly was carried out using SPAdes version
167 3.7 (Bankevich et al. 2012) and contigs were annotated using Prokka 1.11 (Seemann 2014).
6
168 This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the
169 accession SBHZ00000000. Version SBHZ01000000 is used in this paper.
170 Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values
171 between the draft genome sequence of strain TBZ2T (accession number SBHZ00000000) and
172 its closest phylogenetic relatives, P. mendocina ymp (accession number
173 NZ_BBQC00000000) and P. guguanensis JCM 18416T (accession number
174 NZ_FNJJ00000000), were estimated using the GGDC server with the recommended formula
175 2 (Meier-Kolthoff et al. 2013a) and an OrthoANIu algorithm of the ANI Calculator (Lee et
176 al. 2016; Yoon et al. 2017), respectively.
177 The genomic features of the strain TBZ2T were determined after annotation of the draft
178 genome sequence though RAST server (Aziz et al. 2008; Overbeek et al. 2013).
179
180 Results and discussion
181 Strain TBZ2T can grow without NaCl and in the presence of 0.5 to 8.5% of NaCl
182 (optimum 2%). Growth was possible at 10-45 ºC with pH range of 3.0-10.0, optimum growth
183 at 30 °C and pH of 6.0-7.0 in LB (Luria-Bertani). Strain TBZ2T was capable of growing on
184 all three ONR7a solid media supplemented with naphthalene, phenanthrene and anthracene,
185 which means that the bacteria can use PAHs components as the only organic carbon source in
186 medium. TEM image showed that the strain TBZ2T is a motile bacterium harbouring a single
187 polar flagellum with a size of 0.5-0.6 × 1.2-1.7 µm (Fig. S1).
188 The major fatty acids are C18:1 ω7C, C16:1 ω7C/ 15 iso OH, C16:0, C12:0 3-OH, C10:0 3-OH
189 and other phenotypic characteristics of strain TBZ2T are summarized in the species
190 description in Table 1.
191 Analysis of the 16S rRNA gene sequences revealed that strain TBZ2T belonged to the genus
192 Pseudomonas (Fig. 1). The 16S rRNA gene sequence similarities between strain TBZ2T and
7
193 its closely related species were 98.83% with P. oleovorans subsp. oleovorans DSM 1045T
194 (Lee and Chandler 1941; Saha et al. 2010), 98.63% with P. mendocina NBRC 14162T
195 (Palleroni et al.1970), 98.61% with P. oleovorans subsp. lubricantis RS1T (Saha et al. 2010)
196 and 98.49% with P. alcaliphila JCM 10630T (Yumoto et al. 2001) based on EzBioCloud
197 server. However, the pairwise sequence similarities of the 16S rRNA gene calculated
198 following the recommended method of Meier-Kolthoff (Meier-Kolthoff et al. 2013b) showed
199 values of 99.2%, 99.0%, 98.9% and 98.8% between the studied strain and the type strains of
200 P. mendocina, P. oleovorans subsp. lubricantis, Pseudomonas alcaliphila (Yumoto et al.
201 2001) and P. oleovorans subsp. oleovorans, Pseudomonas toyotomiensis (Hirota et al. 2011)
202 and Pseudomonas chengduensis (Tao et al. 2014) respectively (Table 2). These results are not
203 coherent with the distant phylogenetic position of strain TBZ2T from the species cited above.
204 The studied strain formed, in the 16S rRNA gene based-phylogenetic tree, a distinct branch
205 closely related to a subclade encompassing Pseudomonas alcaligenes NBRC 14156T (Monias
206 1928), Pseudomonas guguanensis JCM 18416T (Liu et al. 2013), Pseudomonas
207 pharmacofabricae ZYSR67-ZT (Yu et al. 2018) and Pseudomonas fluvialis ASS-1T (Sudan et
208 al. 2017) (Fig. 1). This clade was located next to the one housing P. alcaliphila, P.
209 mendocina, P. oleovorans subsp. lubricantis and P. oleovorans subsp. oleovorans, P.
210 toyotomiensis and P. chengduensis.
211 Due to this discrepancy in the 16S rRNA gene analyses, three housekeeping genes (rpoD,
212 gyrB and rpoB) known for their significant impact in clarifying the phylogenetic relationship
213 between the closely related species of the genus Pseudomonas were used together with the
214 16S rRNA gene sequences. Strain TBZ2T occupied with high confidence a distinct branch
215 closely related to the type strains of P. mendocina and P. guguanensis in the single genes,
216 rpoD and gyrB, analyses (Fig. S2 and Fig. S3) and MLSA trees (Fig. 2). These results are in
217 line with the phylogenomic tree (Fig. 3) as well as with the lowest genetic distance of 0.004
8
218 and 0.003 detected between strain TBZ2T and P. mendocina and P. guguanensis,
219 respectively.
220 The resultant sequencing data of the genome of strain TBZ2T showed 72 contigs, N50 value
221 of 65559 with 4942 coding sequences and 63 RNAs. The size of the draft genome of the
222 studied strain found to be 5.2 Mb with an in silico G+C content of 64.9% while the genome
223 sequence of P. mendocina NBRC 14162T has a size of 5.1 Mb with 62.8% of G+C content.
224 However, P. guguanensis JCM 18416T has a genome size of 5.08 Mb and G+C content of
225 64.1%. The G+C content of strain TBZ2T was additionally determined by HPLC according to
226 the method of Tóth et al. (2017) which resulted in a value of 65.2 mol%.
227 The dDDH values between the draft genome sequence of strain TBZ2T and its closest
228 relatives, P. mendocina ymp and P. guguanensis JCM 18416T were 25.3% and 26.8%,
229 respectively; values well below the threshold of 70% designed for assigning prokaryotic
230 strains to the same species (Wayne et al. 1987). As shown in Table 2, the dDDH values
231 below the threshold of 70% were obtained between the draft genome sequence of strain
232 TBZ2T and all its relatives with some of which it showed 16S rRNA gene sequences
233 similarity values above the threshold of 98.7% proposed as minimal cutoff for dDDH (Chun
234 et al. 2018).
235 These results are coherent with the ANI values calculated between the draft genome sequence
236 of strain TBZ2T and type strains of P. mendocina ymp (81.5%) and P. guguanensis (79.0%)
237 which found to be below the threshold of 95%-96% for delineation of prokaryotic species
238 (Chun and Rainey 2014; Goris et al. 2007; Richter and Rosselló-Móra 2009).
239
240 Description of Pseudomonas khazarica
241 Pseudomonas khazarica (kha.za.ri.ka N.L. fem. adj. khazarica pertaining to Khazar, a
242 lake in the north of Iran, from where the organism was isolated).
9
243 The isolated bacterium TBZ2T is Gram-negative, rod shaped and motile. The cells
244 produce cream colored colonies on marine agar medium. The shape of the colonies is circular
245 and the angle of the colonies is convex and their edges are entire. The cells can grow without
246 NaCl and tolerate it up to 6.5% after 48 hours and 8.5% after one week with optimum growth
247 in 2% NaCl. This strain is able to grow on MacConkey agar, nutrient agar, brain heart
248 infusion (BHI), Luria-Bertani (LB) media and sea water agar. The bacterium grows at 10-45
249 ºC, pH range of 3.0-10.0 with the efficient growth at 30 °C and pH of 6.0-7.0.
250 The biochemical experiments indicated that the strain TBZ2T is capable of producing
251 oxidase, catalase, amylase, tyrosinase, arginine decarboxylase and phenylalanine deaminase
252 but not able to produce indole, HR2RS and production test of gelatinase is doubtful. Tween 20,
253 Tween 80 and casein hydrolysis and nitrate reduction tests are positive according to lab-made
254 tests. It is positive for reduction of nitrate to nitrite.
255 According to the API 50CH test, acid is produced from glycerol, D-galactose, D-glucose,
256 D-manitol, N-acetylglucosamine, esculin, D-cellobiose, D-maltose, D-lactose (bovine origin),
257 D-saccharose (sucrose) and D-trehalose. But acid is not produced from erythritol, L-
258 arabinose, D-xylose, L-xylose, methyl-βD-xylopyranoside, D-mannose, L-sorbose, L-
259 rhamnose, dulcitol, inositol, D-sorbitol, methyl-αD-mannopyranoside, methyl-αD-
260 glucopyranoside, amygdaline, salicin, D-melibiose inuline, D-melezitose, D-raffinose,
261 amidon (starch), glycogen, D-lyxose, D-tagatose, D-fucose, L- fucose, D-arabitol, L-arabitol,
262 potassium gluconate, potassium 2-ketogluconate and potassium 5-ketogluconate. In API
263 20NE, D-glucose, D-mannose, D-mannitol, potassium gluconate, capric acid, malic acid and
264 trisodium citrate are assimilated, but L-arabinose, N-acetyl-glucosamine, D-maltose, adipic
265 acid and phenylacetic acid are not assimilated. Moreover, in API 20NE and API 20E the
266 strain TBZ2T have enzyme activities for tryptophane deaminase, arginine dihydrolase, urease,
267 lysine decarboxylase and ornithine decarboxylase, but does not display enzyme activities for
10
268 β-glucosidase, N-Acetyl-β-glucosaminidase and β-galactosidase (ortho-nitrophenyl-βD-
269 galactopyranosidase and para-nitrophenyl-βD-galactopyranosidase). Test of gelatinase
270 enzyme is doubtful. The strain TBZ2T is not able to produce indole. Sodium pyruvate
271 acidification is negative. Strain TBZ2T showed a genome size of 5.2 Mb with an in silico
272 G+C content of 64.9 mol% (HPLC, 65.2 mol%).
273 The GenBank accession number for genome and the 16S rRNA gene sequences of strain
274 TBZ2T are SBHZ00000000 and KX712072.3, respectively.
275 The type strain is TBZ2T (=LMG 29674T =KCTC 52410T).
276
277 Acknowledgments
278 This work was supported by the Molecular Medicine Research Center, Tabriz University of
279 Medical Sciences, Tabriz, Iran. We also acknowledge School of Natural and Environmental
280 Sciences, Newcastle University, Newcastle upon Tyne, UK. Genome sequencing was
281 provided by MicrobesNG (http://www.microbesng.uk), which is supported by the BBSRC
282 (grant number BB/L024209/1).
283
284 Compliance with ethical standards
285 Conflict of interest: The authors declare that they have no direct or indirect conflict of
286 interest.
287 Ethical approval: It is the original work of the authors. The work described has not been
288 submitted elsewhere for publication, in whole or in part, and all authors listed carry out the
289 data analysis and manuscript writing and ‘‘This article does not contain any studies with
290 human participants or animals performed by any of the authors’’. Moreover, all authors read
291 and approved the final manuscript.
292 Authors Contributions
11
293 V. T. isolated the bacterium; C. S., S. V. and P. S. did fatty acids, 16S analysis and
294 description; V.T. and V. E. did phenotypic, biochemical and genotypic experiments; I. N.
295 performed the phylogenetic analyses and C. C-A analysed the genome and provided the
296 genome features; M.A. H. and V. T. prepared the manuscript; H.P. K. and M. S. H. wrote and
297 edited the manuscript; V. T., H. P. K. and M.S.H. designed the experiments; H.P.K. and
298 M.S.H. led and supervised the project.
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313 References
12
314 Aziz RK et al. (2008) The RAST Server: rapid annotations using subsystems technology 315 BMC genomics 9:75 316 Bankevich A et al. (2012) SPAdes: a new genome assembly algorithm and its applications to 317 single-cell sequencing Journal of computational biology 19:455-477 318 Beijerinck M Pigments as products of oxidation by bacterial action. In: KNAW, Proceedings, 319 1911. pp 1910-1911 320 Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina 321 sequence data Bioinformatics 30:2114-2120 322 Bosma TN, Middeldorp PJ, Schraa G, Zehnder AJ (1996) Mass transfer limitation of 323 biotransformation: quantifying bioavailability Environmental Science & Technology 324 31:248-252 325 Busquets A, Gomila M, Beiki F, Mulet M, Rahimian H, García-Valdés E, Lalucat J (2017) 326 Pseudomonas caspiana sp. nov., a citrus pathogen in the Pseudomonas syringae 327 phylogenetic group Systematic and applied microbiology 40:266-273 328 Chun J et al. (2018) Proposed minimal standards for the use of genome data for the taxonomy 329 of prokaryotes International journal of systematic and evolutionary microbiology 330 68:461-466 331 Chun J, Rainey FA (2014) Integrating genomics into the taxonomy and systematics of the 332 Bacteria and Archaea International journal of systematic and evolutionary 333 microbiology 64:316-324 334 Corbin DR, Grebenok RJ, Ohnmeiss TE, Greenplate JT, Purcell JP (2001) Expression and 335 chloroplast targeting of cholesterol oxidase in transgenic tobacco plants Plant 336 Physiology 126:1116-1128 337 Farris JS (1972) Estimating phylogenetic trees from distance matrices The American 338 Naturalist 106:645-668 339 Fitch WM (1971) Toward defining the course of evolution: minimum change for a specific 340 tree topology Systematic Biology 20:406-416 341 Fujikawa K, Fort FL, Samejima K, Sakamoto Y (1993) Genotoxic potency in Drosophila 342 melanogaster of selected aromatic amines and polycyclic aromatic hydrocarbons as 343 assayed in the DNA repair test Mutation Research/Fundamental and Molecular 344 Mechanisms of Mutagenesis 290:175-182 345 Gerhardt P (1994) Methods for general and molecular bacteriology. American Society for 346 Microbiology,
13
347 Ghosal D, Ghosh S, Dutta TK, Ahn Y (2016) Corrigendum: Current State of Knowledge in 348 Microbial Degradation of Polycyclic Aromatic Hydrocarbons (PAHs): A Review 349 Frontiers in microbiology 7 350 Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM (2007) 351 DNA–DNA hybridization values and their relationship to whole-genome sequence 352 similarities International journal of systematic and evolutionary microbiology 57:81- 353 91 354 Gregersen T (1978) Rapid method for distinction of Gram-negative from Gram-positive 355 bacteria European Journal of Applied Microbiology and Biotechnology 5:123-127 356 Hirota K, Yamahira K, Nakajima K, Nodasaka Y, Okuyama H, Yumoto I (2011) 357 Pseudomonas toyotomiensis sp. nov., a psychrotolerant facultative alkaliphile that 358 utilizes hydrocarbons International journal of systematic and evolutionary 359 microbiology 61:1842-1848 360 Jin HM, Kim KH, Jeon CO (2015) Alteromonas naphthalenivorans sp. nov., a polycyclic 361 aromatic hydrocarbon-degrading bacterium isolated from tidal-flat sediment 362 International journal of systematic and evolutionary microbiology 65:4208-4214 363 Karlson U, Dwyer D, Hooper S, Moore E, Timmis K, Eltis L (1993) Two independently 364 regulated cytochromes P-450 in a Rhodococcus rhodochrous strain that degrades 2- 365 ethoxyphenol and 4-methoxybenzoate Journal of bacteriology 175:1467-1474 366 Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions 367 through comparative studies of nucleotide sequences Journal of molecular evolution 368 16:111-120 369 Kumari S, Regar RK, Manickam N (2018) Improved polycyclic aromatic hydrocarbon 370 degradation in a crude oil by individual and a consortium of bacteria Bioresource 371 technology 254:174-179 372 Kuykendall L, Roy M, O'neill J, Devine T (1988) Fatty acids, antibiotic resistance, and 373 deoxyribonucleic acid homology groups of Bradyrhizobium japonicum International 374 Journal of Systematic and Evolutionary Microbiology 38:358-361 375 Lee I, Kim YO, Park S-C, Chun J (2016) OrthoANI: an improved algorithm and software for 376 calculating average nucleotide identity International journal of systematic and 377 evolutionary microbiology 66:1100-1103 378 Lee M, Chandler AC (1941) A study of the nature, growth and control of bacteria in cutting 379 compounds Journal of Bacteriology 41:373
14
380 Lefort V, Desper R, Gascuel O (2015) FastME 2.0: a comprehensive, accurate, and fast 381 distance-based phylogeny inference program Molecular biology and evolution 382 32:2798-2800 383 Liu Y-C et al. (2013) Pseudomonas guguanensis sp. nov., a gammaproteobacterium isolated 384 from a hot spring International journal of systematic and evolutionary microbiology 385 63:4591-4598 386 Manickam N, Ghosh A, Jain RK, Mayilraj S (2008) Description of a novel indole-oxidizing 387 bacterium Pseudomonas indoloxydans sp. nov., isolated from a pesticide- 388 contaminated site Systematic and applied microbiology 31:101-107 389 Martins M, Ferreira AM, Costa MH, Costa PM (2016) Comparing the genotoxicity of a 390 potentially carcinogenic and a noncarcinogenic PAH, singly, and in binary 391 combination, on peripheral blood cells of the European sea bass Environmental 392 toxicology 31:1307-1318 393 Meier-Kolthoff JP, Auch AF, Klenk H-P, Göker M (2013a) Genome sequence-based species 394 delimitation with confidence intervals and improved distance functions BMC 395 bioinformatics 14:60 396 Meier-Kolthoff JP, Göker M (2019) TYGS is an automated high-throughput platform for 397 state-of-the-art genome-based taxonomy Nature Communications 10:2182 398 doi:10.1038/s41467-019-10210-3 399 Meier-Kolthoff JP, Göker M, Spröer C, Klenk H-P (2013b) When should a DDH experiment 400 be mandatory in microbial taxonomy? Archives of Microbiology 195:413-418 401 Miller LT (1982) Single derivatization method for routine analysis of bacterial whole-cell 402 fatty acid methyl esters, including hydroxy acids Journal of Clinical Microbiology 403 16:584-586 404 Monias BL (1928) Classification of bacterium alcaligenes, pyocyaneum, and fluorescens The 405 Journal of Infectious Diseases:330-334 406 O’Mahony MM, Dobson AD, Barnes JD, Singleton I (2006) The use of ozone in the 407 remediation of polycyclic aromatic hydrocarbon contaminated soil Chemosphere 408 63:307-314 409 Overbeek R et al. (2013) The SEED and the Rapid Annotation of microbial genomes using 410 Subsystems Technology (RAST) Nucleic acids research 42:D206-D214 411 Palleroni N (1984) Genus I. Pseudomonas Bergey's manual of systematic bacteriology 1:141- 412 199
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413 Richter M, Rosselló-Móra R (2009) Shifting the genomic gold standard for the prokaryotic 414 species definition Proceedings of the National Academy of Sciences 106:19126- 415 19131 416 Saha R, Spröer C, Beck B, Bagley S (2010) Pseudomonas oleovorans subsp. lubricantis 417 subsp. nov., and reclassification of Pseudomonas pseudoalcaligenes ATCC 17440T as 418 later synonym of Pseudomonas oleovorans ATCC 8062T Current microbiology 419 60:294-300 420 Seemann T (2014) Prokka: rapid prokaryotic genome annotation Bioinformatics 30:2068- 421 2069 422 Stanier RY, Palleroni NJ, Doudoroff M (1966) The aerobic pseudomonads a taxonomic study 423 Microbiology 43:159-271 424 Sudan SK et al. (2017) Pseudomonas fluvialis sp. nov., a novel member of the genus 425 Pseudomonas isolated from the river Ganges, India International journal of systematic 426 and evolutionary microbiology 68:402-408 427 Tao Y, Zhou Y, He X, Hu X, Li D (2014) Pseudomonas chengduensis sp. nov., isolated from 428 landfill leachate International journal of systematic and evolutionary microbiology 429 64:95-100 430 Tarhriz V et al. (2014) Isolation and characterization of naphthalene-degradation bacteria 431 from Qurugol Lake Located at Azerbaijan Biosci Biotechnol Res Asia 11:715 432 Tarhriz V, Mohammadzadeh F, Hejazi MS, Nematzadeh G, Rahimi E ((2011)) Isolation and 433 characterization of some aquatic bacteria from Qurugol Lake in Azerbaijan under 434 aerobic conditions Advances in Environmental Biology:3173-3179 435 Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of 436 progressive multiple sequence alignment through sequence weighting, position- 437 specific gap penalties and weight matrix choice Nucleic acids research 22:4673-4680 438 Tóth E, Szuróczki S, Kéki Z, Kosztik J, Makk J, Boka K, Spröer C, Márialigeti K, Schumann 439 P.(2017). Brevundimonas balnearis sp. nov., isolated from the well water of a thermal 440 bath. Int J Syst Evol Microbiol 67:1033-1038. 441 Waturangi DE, Francisca I, Susanto CO (2011) Genetic diversity of methylotrophic bacteria 442 from human mouth based on amplified ribosomal DNA restriction analysis (ARDRA) 443 HAYATI Journal of Biosciences 18:77-81
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444 Wayne L et al. (1987) Report of the ad hoc committee on reconciliation of approaches to 445 bacterial systematics International Journal of Systematic and Evolutionary 446 Microbiology 37:463-464 447 Wilson SC, Jones KC (1993) Bioremediation of soil contaminated with polynuclear aromatic 448 hydrocarbons (PAHs): a review Environmental pollution 81:229-249 449 Yoon S-H, Ha S-m, Lim J, Kwon S, Chun J (2017) A large-scale evaluation of algorithms to 450 calculate average nucleotide identity Antonie van Leeuwenhoek 110:1281-1286 451 Yu X-Y, Zhai J-Y, Wu C, Zhang C-Y, Shi J-Y, Ding L-X, Wu M (2018) Pseudomonas 452 pharmafabricae sp. nov., isolated from pharmaceutical wastewater Current 453 microbiology 75:1119-1125 454 Yumoto I et al. (2001) Pseudomonas alcaliphila sp. nov., a novel facultatively psychrophilic 455 alkaliphile isolated from seawater International journal of systematic and evolutionary 456 microbiology 51:349-355
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474 Table and Figures Legends:
475 Table 1 Comparison of the characteristics of Pseudomonas khazarica strain TBZ2T and its
476 closely related species including Pseudomonas species.
477 Taxa: 1, Pseudomonas khazarica TBZ2T; 2, Pseudomonas oleovorans subsp. oleovorans
478 DSM 1045T; 3, Pseudomonas oleovorans subsp. lubricantis RS1T; 4, Pseudomonas
479 mendocina BCRC 10458T; 5, Pseudomonas indoloxydans IPL-1T; 6, Pseudomonas
480 alcaliphila AL15-21T. Data in column 4-6 were obtained from Manickam and colleagues
481 report (Manickam et al. 2008).
482 Table 2. dDDH and pairwise 16S rRNA gene sequence similarity between strain TBZ2T and
483 closest phylogenetic neighbors.
484 Fig. 1 Maximum-likelihood phylogenetic tree derived from 16S rRNA gene sequence data
485 showing the phylogenetic relationships between Pseudomonas khazarica sp. nov. TBZ2T and
486 its related taxa. Numbers above the nodes correspond to the support values for Maximum-likelihood
487 (left) and Maximum-parsimony (right).
488 Fig. 2 Maximum-likelihood MLSA phylogenetic tree based on concatenated sequences of
489 16S rRNA, rpoD, gyrB and rpoB genes and showing the phylogenetic relationships between
490 Pseudomonas khazarica sp. nov. TBZ2T and its related taxa. Numbers above the nodes
491 correspond to the support values for Maximum-likelihood (left) and Maximum-parsimony (right).
492 Fig. 3. Tree inferred with FastME 2.1.6.1 (Lefort et al. 2015) from GBDP distances 493 calculated from genome sequences. The branch lengths arescaled in terms of GBDP distance
494 formula d5. The numbers above branches are GBDP pseudo-bootstrap support values from 495 100 replications, with an average branch support of 81.1 %. The tree was rooted at the 496 midpoint (Farris 1972) (Farris 1972). 18
497
498 Fig. S1 TEM image of TBZ2T.
499 Fig S2 Maximum-likelihood phylogenetic tree derived from rpoD gene sequences showing
500 the phylogenetic relationships between Pseudomonas khazarica sp. nov. TBZ2T and its
501 related taxa. Numbers above the nodes correspond to the support values for Maximum-likelihood
502 (left) and Maximum-parsimony (right).
503 Fig. S3 Maximum-likelihood phylogenetic tree derived from gyrB gene sequences showing
504 the phylogenetic relationships between Pseudomonas khazarica sp. nov. TBZ2T and its
505 related taxa. Numbers above the nodes correspond to the support values for Maximum-likelihood
506 (left) and Maximum-parsimony (right).
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524 525 Table 1 526 527 Characteristic 1 2 3 4 5 6 Motility + + + + + + 528 Nitrate reduction + - - - + + Growth at: 529 4 °C - - - - - + 10 °C + - - - + + 530 45°C + - + - - - pH 10 + - + - - + 531 Growth in: NaCl concentration range (w/v) (%) 0.0 - 8.5 0.0 - 3.0 0.0 - 5.0 0.0-7.0 2.5 - 5.0 3.0 - 5.0 532 Hydrolysis of: Casein - - - - - + 533 Tween 20 + - - + + + Tween80 + + + + + + 534 Starch + - - - - - aesculin + - - - - - 535 Production of: Indole ------536 Acetoin (Voges–Proskauer test) ------H2S ------537 Enzymes: Urease + - + - - - 538 Gelatinase - - - - + Arginine dihydrolase + - - + - + 539 Ornithine decarboxylase + + + + + + Acid production from: 540 D-Glucose + - - + - + D-Fructose + + - + + + 541 D-Maltose + - - - + - D-Xylose - - - + + - 542 D-Mannose - - - + + - L-Arabinose - - - + + - 543 Cellobiose + + - + + - Raffinose - + - - - - 544 Sugar Utilization D-Glucose + + - + - + 545 L-Arabinose - + - + + + D-Mannose + - - - - 546 D-Fructose - + - - - - D-Xylose - - - + 547 D-mannitol + - - - - + Utilization of: 548 L-Arginine + - - + - + L-Ornithine + - + - - - 549 Trisodium citrate + + + + + + Pyrovate - + - + + + Polycyclic aromatic hydrocarbons 550 + ? ? +[1] ? +[2] (PAHs) utilization 551 DNA G+C content (mol%, HPLC) 65.2 62.2 63.5 63.8 67.2 63.2 552 553 [?]: We didn’t find any reports regarding utilization of PAHs by these strains. [1], (Kumari et al. 2018), [2], 554 (O’Mahony et al. 2006).
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563 Table 2
1 2 3 4 5 6 7 (%) 1 Strain TBZ2T - 25.3 19.8 26.2 24.1 30.7 28.6 2 Pseudomonas mendocina NBRC 14162T 99.2 ------3 Pseudomonas oleovorans subsp. lubricantis RS1T 99.0 ------4 Pseudomonas alcaliphila JCM 10630T 98.9 ------5 Pseudomonas oleovorans subsp. oleovorans DSM 98.8 ------1045T 6 Pseudomonas toyotomiensis HT-3T 98.8 ------7 Pseudomonas chengduensis MBRT 98.8 ------564
565 The values on bold correspond to the dDDH while the non-bold ones, displayed on column 1, are the pairwise 566 16S rRNA gene sequence similarity (%). 567
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570 571 Fig. 1
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592 Fig. S1 593
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597 Fig. S2
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599 600 601 Fig. S3
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