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1 Pseudomonas versuta sp. nov., isolated from Antarctic soil 1 2 3 1,2 3 1 2,4 1,5 4 2 Wah Seng See-Too , Sergio Salazar , Robson Ee , Peter Convey , Kok-Gan Chan , 5 6 3 Álvaro Peix 3,6* 7 8 4 1Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of 9 10 11 5 Science University of Malaya, 50603 Kuala Lumpur, Malaysia 12 13 6 2National Antarctic Research Centre (NARC), Institute of Postgraduate Studies, University of 14 15 16 7 Malaya, 50603 Kuala Lumpur, Malaysia 17 18 8 3Instituto de Recursos Naturales y Agrobiología. IRNASA -CSIC, Salamanca, Spain 19 20 4 21 9 British Antarctic Survey, NERC, High Cross, Madingley Road, Cambridge CB3 OET, UK 22 23 10 5UM Omics Centre, University of Malaya, Kuala Lumpur, Malaysia 24 25 11 6Unidad Asociada Grupo de Interacción Planta-Microorganismo Universidad de Salamanca- 26 27 28 12 IRNASA ( CSIC) 29 30 13 , IRNASA-CSIC, 31 32 33 14 c/Cordel de Merinas 40 -52, 37008 Salamanca, Spain. Tel.: +34 923219606. 34 35 15 E-mail address: [email protected] (A. Peix) 36 37 38 39 16 Abstract: 40 41 42 43 17 In this study w e used a polyphas ic taxonomy approach to analyse three bacterial strains 44 45 18 coded L10.10 T, A4R1.5 and A4R1.12 , isolated in the course of a study of quorum -quenching 46 47 19 bacteria occurring Antarctic soil . The 16S rRNA gene sequence was identical in the three 48 49 50 20 strains and showed 99.7% pairwise similarity with respect to the closest related species 51 52 21 Pseudomonas weihenstephanensis WS4993 T, and the next closest related species were P. 53 54 T T T 55 22 deceptionensis M1 (99.5%), P. psychrophila E-3 , P. endophytica BSTT44 and P. fragi 56 57 23 ATCC 4973 T (99. 2%) . Therefore, the three strains were classified within the genus 58 59 60 24 Pseudomonas . Analysis of housekeeping gene s (rpoB , rpoD and gyrB ) sequences showed 61 62 63 64 65 25 similarities of 84-95% with respect to the closest relatives, confirming its phylogenetic 1 2 26 affiliation. The whole genome average nucleotide identity ( ANI) values were more than 99% 3 4 5 27 similar among the three strains, and less than 86% to the closest related species type strains . 6 7 28 The respiratory quinone is Q9. The major fatty acids are C16:0, C16:1 ω7c/ C16:1 ω6c in 8 9 10 29 summed feature 3 and C18:1 ω7c / C18:1 ω6c in summed feature 8 . The strain s are oxidase - 11 12 30 and catalase -positive. The arginine dihydrolase and urease tests are positive . Growth occur s 13 14 15 31 at 4 –30 C with an optimum at 28 C, and at pH 4.0 –10 . The DNA G+C content is 58. 2-58.3 16 17 32 mol % . The combined genotypic, phenotypic and chemotaxonomic data support the 18 19 T 20 33 classification of strain s L10.10 , A4R1.5 and A4R1.12 into a novel species of Pseudomonas , 21 T T 22 34 for which the name P. versuta sp. nov. is proposed. The type strain is L10.10 (LMG 29628 , 23 24 35 DSM 101070 T). 25 26 27 36 28 29 37 Keywords: Pseudomonas, taxonomy , Antarctica, Antarctic soil, quorum quenching, quorum 30 31 32 38 sensing 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 39 The genus Pseudomonas was first described by Migula [29] and is one of the most commonly 1 2 40 reported bacteria in Antarctica [28]. To date, according to the List of Prokaryotic Names with 3 4 5 41 Standing in Nomenclature (http://www.bacterio.net/pseudomonas.html) , there are more than 6 7 42 150 species described in this genus. Pseudomonas is one of the most diverse and ubiquitous 8 9 10 43 bacterial genera, and representatives have been isolated worldwide including from many 11 12 44 extreme environments such as Antarctica [37], given their extraordinarily versatile 13 14 45 metabolism. Even though psychrophilic strains of P. aeruginosa, P. fluorescens, P. putida 15 16 17 46 and P. syringae were reported by Shivaji et al. [47] in Antarctic soil and water samples, 18 19 47 entirely novel species of Pseudomonas from Antarctica were first described by Reddy et al. 20 21 22 48 in 2004 with the isolation of P. antarctica, P. proteolytica and P. meridiana from 23 24 49 cyanobacterial mat samples [39] . Since then, six further novel species have been isolated 25 26 27 50 from Antarctic or sub-Antarctic regions, including P. guineae [5], P. pelagia [18], P. 28 29 51 extremaustralis [27], P. deceptionensis [6], P. prosekii [23], and the most recently described 30 31 32 52 P. yamanorum [4], isolated from the Isla de los Estados (Tierra del Fuego, Argentina). 33 34 35 53 The production of N- acyl-homoserine lactones (AHLs) mediating quorum sensing (QS) is a 36 37 38 54 common property identified in numerous Pseudomonas strains for the regulation of a variety 39 40 55 of phenotypes in relation to the population density [24, 53, 55]. Certain Pseudomonas species 41 42 43 56 were also found to possess AHL acylase activity, an enzyme that can degrade AHLs, 44 45 57 allowing the utilization of this compound as nitrogen and energy sources [17, 26, 49]. 46 47 58 Degradation of QS signaling is termed as quorum quenching (QQ), and has been suggested as 48 49 50 59 a highly promising alternative approach for infection control since QQ does not affect 51 52 60 bacterial viability and could minimize the selective pressure to develop resistance [14]. QQ 53 54 55 61 enzyme production has been widely described in mesophilic bacterial strains [12, 14]. In this 56 57 62 context, isolation of novel psychrotolerant QQ bacteria will help to unravel the potential of 58 59 60 61 62 63 64 65 63 QQ bacteria as biocontrol agents in cold environments at high latitudes or in temperate 1 2 64 regions. 3 4 5 6 65 During an ecological survey of QQ soil bacteria in various locations in Antarctica (Fig. 1), 7 8 66 we isolated three strains from soil samples using QQ bacteria enrichment medium [8]. The 9 10 11 67 soil samples were collected from Lagoon Island and Anchorage Island (Ryder Bay, Adelaide 12 13 68 Island) and taken to the laboratory for bacterial isolation. In brief, 1 g of soil sample and 5 ml 14 15 16 69 sterile QQ bacteria enrichment medium containing 100 µg synthetic C 6-HSL as sole carbon 17 18 70 source, were mixed in a sterile 50 ml plastic tube and incubated at 4 °C and 150 rpm. After 1 19 20 21 71 week of incubation, 100 µl of the bacterial suspension were added into new QQ bacteria 22 23 72 enrichment medium again including C 6-HSL. This step was repeated three times and finally, 24 25 73 100 µl of bacterial suspension were plated onto Luria-Bertani (LB) agar. The three strains 26 27 T 28 74 obtained, coded as L10.10 , A4R1.5 and A4R1.12, were subjected to a polyphasic taxonomic 29 30 75 study, and classified into the genus Pseudomonas within the Class Gammaproteobacteria 31 32 33 76 according to 16S rRNA and housekeeping gene analyses . The genomic, phylogenetic, 34 35 77 chemotaxonomic and phenotypic data obtained showed that they represent a novel species for 36 37 38 78 which the name Pseudomonas versuta sp. nov. is proposed. 39 40 41 79 In order to analyse genetic variability and to assess for clonality, the three strains were 42 43 44 80 j P PD f m M13 (5’ - GAGGGTGGCGGTTCT 45 46 81 –3’) v [41]. The bands present in each profile were coded for input into 47 48 49 82 a database including all the strains studied and Dice similarity coeffi cient was calculated to 50 51 83 construct the distance matrix. A dendrogram was constructed from the distance matrix using 52 53 54 84 the unweighted pair group with arithmetic mean (UPGMA) , using the GelCompar II program 55 56 85 from Bionumerics platform (Applied Maths NV). The RAPD patterns obtained showed that 57 58 86 the three strains L10.10 T, A4R1.5 and A4R1.12 are not clones since they present different 59 60 61 62 63 64 65 87 RAPD profiles. Strains L10.10 T and A4R1.5 show less than 90% similarity between them 1 2 88 and less tha n 80% with respect to the strain A4R1.12 in the Dice dendrogram, presenting a 3 4 5 89 high genetic variability with respect to the closely related Pseudomonas species (Fig. S1). 6 7 8 90 Colony morphologies of the three strains were observed after 48 h of incubation on LB agar. 9 10 11 91 Gram-staining was conducted using a Difco Gram stain set followed by observation under a 12 13 92 Leica DM750 microscope (Leica Microsystems). Type of flagellation and cell morphology 14 15 16 93 were documented using a Hitachi TM3030 scanning electron microscope and a scanning 17 18 94 transmission electron microscope (STEM, LIBRA 120; Carl Zeiss AG, Germany). Briefly, 19 20 21 95 cells were grown on LB broth overnight at 25 C, harvested by centrifugation and 22 23 96 resuspended in PBS. The overnight suspension cells were then stained using 1% 24 25 26 97 phosphotungstic acid on a Formvar grid and observed at an operating voltage of 80 kV. 27 28 98 Strain L10.10 T is Gram negative, rod-shaped (0.6-0.8 x 1.8-2.3µm) and motile by one polar- 29 30 99 subpolar flagellum (Fig. S2). 31 32 33 34 100 The complete genome of strain L10.10 T has been previously sequenced, enabling the 35 36 37 101 discovery of various genes encoding for plant growth promoting properties and plant disease 38 39 102 prevention attributes [45]. The draft genomes of strains A4R1.5 and A4R1.12 were obtained 40 41 103 in this study in order to perform whole genome sequence analysis. The genomic DNA was 42 43 44 104 extracted using the MasterPure DNA purification kit (Epicenter, USA) following the 45 46 105 mf’ . T m w the Nextera DNA 47 48 49 106 Library prepation kit (Illumina, USA) and subsequently subjected to sequencing using an 50 51 107 Illumina MiSeq sequencer. The reads generated were filtered and assembled using CLC 52 53 54 108 genomic benchwork. 55 56 57 109 In order to establish the phylogenetic affiliation of the novel isolates, the complete 16S rRNA 58 59 T 60 110 gene sequences of strains L10.10 , (1466 bp), A4R1.5 (1525 bp) and A4R1.12 (1525 bp) 61 62 63 64 65 111 were retrieved from the complete genome sequence of L10.10 T and draft genome sequences 1 2 112 from A4R1.5 and A4R1.12, and compared with those held in databases using Ez-Taxon-e 3 4 5 113 [19] and BlastN [3]. Pairwise similarity analysis demonstrated that the three strains showed 6 7 114 100% sequence similarity among them, and therefore only strain L10.10 T was included in the 8 9 T 10 115 16S rRNA gene phylogenetic analysis. Comparison of strain L10.10 sequence against the 11 12 116 databases mentioned showed that it should be classified into the genus Pseudomonas . The 13 14 117 closest related species is P. weihenstephanensis WS4993 T [54] with 99.7% pairwise 15 16 T 17 118 similarity, and the following closest related species are P. deceptionensis M1 [6] with 18 19 119 99.5%, P. psychrophila E-3 T [57], P. endophytica BSTT44 T [38] and P. fragi ATCC 4973 T 20 21 T T 22 120 [15] with 99.2%, P. helleri WS4917 [54] with 99.0%, P. taetrolens IAM1653 [16] with 23 24 121 98.9% and P. lundensis ATCC 49968 T [30] with 98.6% sequence similarity. For 16S rRNA 25 26 27 122 gene phylogenetic analysis, the type strains of the closely related Pseudomonas species 28 29 123 showing more than 98% sequence similarity with respect to strain L10.10 T were included. 30 31 32 124 Alignment was performed using the Clustal_X software [52]. The distances were calculated 33 34 125 according to Kimura´s two-parameter model [20]. Phylogenetic trees of the 16S rRNA gene 35 36 126 were inferred using the neighbour-joining analysis (NJ) [43] and maximum likelihood (ML) 37 38 39 127 [42]. All the analyses were performed using MEGA 6.06 software [51]. As can be seen in the 40 41 128 ML phylogenetic tree (Fig. 2), the strain L10.10 T clustered separately in a group that included 42 43 44 129 the closest related species P. weihenstephanensis and P. deceptionensis , branching in a wider 45 46 130 group formed by the closest species mentioned above. The results were congruent with the 47 48 49 131 tree topology obtained after NJ phylogenetic analysis (data not shown). 50 51 52 132 To establish precisely the phylogenetic affiliation of the novel isolates, in addition to the 16S 53 54 55 133 rRNA gene sequence analysis, we also studied the three housekeeping genes more commonly 56 57 134 used in the phylogenetic analysis of Pseudomonas species, rpoD, rpoB and gyrB genes [1, 58 59 60 135 22, 31, 32, 33, 38, 54]. The phylogenetic analysis of the concatenated rpoD, rpoB and gyrB 61 62 63 64 65 136 genes sequences was performed with the same methodology as for the 16S rRNA gene and 1 2 137 showed that the strains L10.10 T, A4R1.5 and A4R1.12 clustered in a separate branch related 3 4 5 138 to a group formed by the type strains of the closely related species P. weihenstephanensis, P. 6 7 139 deceptionensis, P. psychrophila and P. fragi (Fig. 3). As can be seen in the ML phylogenetic 8 9 10 140 tree (Fig. 3), all these strains are located in a wider group including also the species P. 11 12 141 lundensis, P. taetrolens, P. helleri and P. endophytica, confirming the results obtained from 13 14 142 the phylogenetic analysis of the 16S rRNA gene sequence. This group “ P. 15 16 17 143 fragi ” w “ P. fluorescens ” f Pseudomonas species defined by Mulet 18 19 144 et al. [33] after the phylogenetic analysis of the concatenated 16S rRNA gene and the three 20 21 22 145 mentioned housekeeping genes. The same topologies were again obtained after NJ 23 24 146 phylogenetic analysis (data not shown). The identities of the housekeeping genes sequences 25 26 27 147 of the novel isolates with respect to the closely related species ‘ P. fragi 28 29 148 subgroup ’ ranged from 84.7-95.6% for rpoD , 89.7-95.2% for rpoB , and 88.9-93.1% in the 30 31 32 149 case of gyrB gene. These values are in the range of those found for other species of the genus 33 34 150 Pseudomonas [38], and support the classification of the novel isolates within the genus 35 36 151 Pseudomonas , representing an as yet undescribed species of this genus. 37 38 39 40 152 In addition to the 16S rRNA gene sequence and MLSA analyses, in order to provide support 41 42 43 153 for the classification of these Antarctic strains as a novel species, we carried out whole 44 45 154 genome sequence analysis considering the same closely related species included in the 46 47 155 previous phylogenetic analyses described above. Generally, an average nucleotide identity 48 49 50 156 (ANI) value of 95-96 % has been accepted as the cutoff threshold for bacterial species 51 52 157 delineation corresponding to 70 % DNA relatedness [13, 40]. ANI analysis was performed 53 54 55 158 using the orthologous average nucleotide identity tool, OAT [25]. According to the data 56 57 159 obtained, the strain s L10.10 T, A4R1.5 and A4R1.12 showed more than 99% ANI similarity 58 59 60 160 among them, and less than 86% ANI similarity values with respect to all the close st related 61 62 63 64 65 161 species (Table S1), confirming that the three strains belong to the same species, which should 1 2 162 be classified as novel species of the genus Pseudomonas when the range threshold values of 3 4 5 163 94-96 % ANI similarity proposed by Richter and Rossello-Mora [40] for delineation of 6 7 164 bacterial species is considered. 8 9 10 165 11 12 166 A genomic comparison across the L10.10 T, A4R1.5 and A4R1.12 genomes and their close 13 14 167 relatives ‘ P. fragi ’ at nucleotide level was carried out using the 15 16 T 17 168 BLAST Ring Image Generator (BRIG) [2]. The genome of strain L10.10 was used as the 18 19 169 reference genome. The genome sequence accession numbers for L10.10 T, A4R1.5 and 20 21 22 170 A4R1.12 are CP012676, MPJC00000000 and MPJD00000000, respectively. The genome 23 24 171 sequence accession numbers for the closely related species are P. weihenstephanensis , 25 26 27 172 JYLF01000000; P. taetrolens , JYLA00000000.; P. psychrophila , JYKZ01000000; P. 28 29 173 lundensis , JYKY00000000; P. helleri , JYLD00000000; P. fragi , AHZX00000000; P. 30 31 32 174 deceptionensis , JYKX00000000; and P. endophytica , LLWH00000000. The BRIG analysis 33 34 175 indicated that most regions within the analyzed genomes were conserved with at least 70 % 35 36 176 or greater similarity (Fig. S3a). However, strains L10.10 T, A4R1.5 and A4R1.12 showed 37 38 39 177 higher similarity with each other, with most regions being conserved (Fig. S3b). The pan- and 40 41 178 core-genome analysis were performed using the ultra-fast computational pipeline BPGA 42 43 44 179 (Bacterial Pan Genome Analysis tool) [7]. The analysis indicates that all the genomes 45 46 180 considered share 2669 common genes, representing about half of the genes in each genome. 47 48 T 49 181 In the genome of L10.10 there are 10 unique genes compared to other genomes tested. A 50 51 182 summary of the core- and pan-genome analyses is given in Table S2. 52 53 54 T 55 183 G+C content of strain L10.10 was 58.2 mol % as determined from the complete genome 56 57 184 sequence, and ranged between 58.2-58.3 mol % from the draft genome sequences for the 58 59 60 61 62 63 64 65 185 strains A4R1.5 and A4R1.12. These values are within the range commonly found in 1 2 186 Pseudomonas species. 3 4 5 6 187 The cellular fatty acids were analyzed by using the Microbial Identification System (MIDI; 7 8 188 Microbial ID) Sherlock 6.1 and the library RTSBA6, according to the technical instructions 9 10 T 11 189 provided by this system [44]. Strain L10.10 and the type strains of the same closely related 12 13 190 species m ‘ P. fragi ’ 14 15 16 191 MLSA analysis were grown on TSA plates (Becton Dikinson, BBL) for 24 h at 28°C and 17 18 192 harvested in late log growth phase. The major fatty acids of strain L10.10 T were 16:0 19 20 21 193 (29.4%), C16:1 ω7c/ C16:1 ω6c in summed feature 3 (35.8%) and C18:1 ω7c / C18:1 ω6c in 22 23 194 summed feature 8 (11.5%). As expected, all the relatives clustering in the same phylogenetic 24 25 195 group with strain L10.10 T shared similar fatty acid profiles (Table 1). L10.10 T had the three 26 27 28 196 fatty acids typically present in the genus Pseudomonas according to Palleroni [35], which are 29 30 197 C10:0 3OH, C12:0 and C12:0 3OH. 31 32 33 198 34 35 199 The strain L10.10 T was cultivated for 24 h on TSA plates (Becton Dikinson, BBL) at 28°C to 36 37 38 200 obtain the cell mass required for quinone analysis, which was carried out from freeze-dried 39 40 201 cells as described by Collins and Jones [11], Tamaoka et al. [50] and Collins [10]. The strain 41 42 T 43 202 L10.10 contained Q9 as the main respiratory quinone, which is also typical in species of the 44 45 203 genus Pseudomonas [36]. 46 47 204 48 49 50 205 Physiological and biochemical tests were performed as previously described [37] including 51 52 206 for comparison the type strains of the same Pseudomonas species chosen for FAME analysis 53 54 55 207 and whole genome comparison analysis. For fluorescent pigment analysis, cells were grown 56 57 208 in King B broth and tested for pigment production [21]. Strains L10.10 T, A4R1.5 and 58 59 60 209 A4R1.12 did not produce a fluorescent pigment in this medium. Catalase activity was 61 62 63 64 65 210 assayed using 3% (v/v) H 2O2 and determined by assessing the production of copious bubbles, 1 2 211 and oxidase activity was determined using 1% (w/v) N,N,N’, N’,-tetramethyl 1,4- 3 4 5 212 phenylenediamine (bioMérieux) as described by Smibert & Krieg [48]. Positive reactions 6 7 213 were observed in both tests for strains L10.10 T, A4R1.5 and A4R1.12. Additionally API 8 9 10 214 20NE (bioMérieux) and Biolog GN2 Microplates (Biolog, Inc., Hayward, Calif.) were used 11 12 215 fw mf’ . T f P 20E w f 48 h 13 14 216 incubation at 28ºC. 15 16 17 217 18 19 218 Phenotypic characteristics of the new species are reported below in the species description, 20 21 22 219 and the differences with respect to the closest related Pseudomonas species and the type 23 24 220 species of the genus, P. aeruginosa , are recorded in Table 2. The phenotypic characteristics 25 26 T 27 221 of strains L10.10 A4R1.5 and A4R1.12 provide further support for their classification as a 28 29 222 novel species of Pseudomonas , since they are motile Gram-negative strictly aerobic rods, 30 31 32 223 catalase- and oxidase-positive and motile by a polar flagellum [46]. These three strains can be 33 34 224 differentiated from other species of the genus by their 16S rRNA and housekeeping gene 35 36 225 sequences, ANI values, genome nucleotide analysis, and their phenotypic and 37 38 39 226 chemotaxonomic characteristics, and therefore they should be classified as a novel species of 40 41 227 Pseudomonas , for which we propose the name Pseudomonas versuta sp. nov. 42 43 44 228 45 46 47 229 Description of Pseudomonas versuta sp. nov. 48 49 50 51 230 Pseudomonas versuta (v.’. L. fm. j. v , adroit, shrewd, ingenious, referring to 52 53 54 231 the ability for quorum-quenching activity). 55 56 232 Strictly aerobic, Gram-stain-negative, non-spore forming straight rods of 1.8-2.3 µm in 57 58 233 length and 0.6-0.8 µm in diameter, motile by a polar flagellum, occurring singly and in pairs. 59 60 61 62 63 64 65 234 Colonies are 1.5 –2.0 mm in diameter, raised, circular-shaped, and cream-colored after 48 h 1 2 235 incubation at 25 C on LB agar. Growth occurs at 4 –30 C with an optimum at 28 C, and at 3 4 5 236 pH 4.0 –10.0. Tolerant to 0 –7% NaCl (w/v) in LB broth. Strictly aerobic with oxidative 6 7 237 metabolism and no fermentation of sugars in peptone media. No diffusible fluorescent 8 9 10 238 pigment is produced on King B medium. The major respiratory ubiquinone is Q9. Major fatty 11 12 239 acids are 16:0, 16:1 ω7c/ 16:1 ω6c in summed feature 3 and 18:1 ω7c/18:1 ω6c in summed 13 14 15 240 feature 8. Oxidase- and catalase-positive. In API 20 NE system arginine dihydrolase and 16 17 241 urease are positive. Indole and ß –galactosidase production as well as nitrate reduction and 18 19 20 242 esculine hydrolysis are negative. Assimilation of glucose, arabinose, mannose, mannitol, 21 22 243 gluconate, caprate, malate and citrate is positive. Negative results were obtained for 23 24 244 assimilation of maltose, adipate and phenylacetate. Assimilation of N-acetyl-glucosamine is 25 26 27 245 weak. In Biolog GN2 plates the assimilation of tween 40, tween80, L-arabinose, D-arabitol, 28 29 246 D-fructose, L-fucose, D-galactose, α-D-glucose, m-inositol, D-mannitol, D-mannose, D- 30 31 32 247 sorbitol, sucrose, D-trehalose, methyl-piruvate, acetate, cis-aconitate, citrate, D-galactonate 33 34 248 lactone, D-galacturonate, D-gluconate, D-glucosaminate, D-glucuronate, -hydroxy-butyrate, 35 36 37 249 α-ketoglutarate, D, L-lactate, propionate, quinate, D-saccharate, bromo-succinate, 38 39 250 glucuronamide, D-alanine, L-alanine, L-alanyl-glycine, L-asparagine, L-aspartate, L- 40 41 42 251 glutamate, glycyl-L-glutamate, L-histidine, L-leucine, L-ornithine, L-proline, L- 43 44 252 pyroglutamate, D-serine, L-serine, D,L , γ -aminobutyrate, urocanate, inosine, 45 46 253 uridine, putrescine, 2-aminoethanol and glycerol is positive. Negative results were obtained 47 48 49 254 f α -cyclodextrin, dextrin, glycogen, N-acetyl-D-galactosamine, adonitol, D-cellobiose, i- 50 51 255 erythritol, gentiobiose, α-D-lactose, lactulose, maltose, D-melibiose, -methyl-D-glucoside, 52 53 54 256 D-psicose, D-raffinose, L-rhamnose, turanose, xylitol, formate, α-hydroxybutirate, γ- 55 56 257 hydroxybutyrate, , α -k, α -ketovalerate, malonate, sebacate, glycyl-L- 57 58 59 258 aspartate, L-threonine, thymidine, phenylethylamine, 2,3-butanediol and glucose-6- 60 61 62 63 64 65 259 phosphate. Finally, assimilation of N-acetyl-D-glucosamine, L-phenylalanine, and D,L- α- 1 2 260 glycerolphosphate was weak. Variable results were observed for assimilation of succinate, 3 4 5 261 mono-methyl-succinate, L-alaninamide, succinamate, p-hydroxyphenylacetate, hydroxy-L- 6 7 262 proline and glucose-1-phosphate. G+C base composition was 58.2-58.3 mol%. The type 8 9 T T T 10 263 strain is L10.10 (LMG 29628 , DSM 101070 ), isolated from soil obtained from Lagoon 11 12 264 Island, Antarctica. Digital Protologue Taxonumber: TA00067 13 14 265 15 16 17 18 266 Acknowledgements 19 20 21 22 267 We are grateful to Mareike Wenning for kindly providing the type strains of P. 23 24 268 weihenstephanensis and P. helleri , and also to Oleg Loginov, Martha H. Ramírez -Bahena and 25 26 27 269 Zuleica García Palacios for help and support . This work was supported by the University of 28 29 270 Malaya (UM) Hig h Impact Research Grants (UM -MOHE HIR Grant 30 31 32 271 UM.C/625/1/HIR/MOHE/CHAN/14/1, Grant No. H -50001 -A000027; UM -MOHE HIR 33 34 272 Grant UM.C/625/1/HIR/MOHE/CHAN/01, Grant No. A -000001 -50001) awarded to Kok - 35 36 273 Gan Chan. Wah -Seng See -Too thanks to Brigh t Sparks Program of Univ ersity of Malaya for 37 38 39 274 the scholarship. Fieldwork in Antarctica was supported by the British Antarctic Survey. 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Extremophiles 5,343 –349 51 52 507 53 54 55 508 56 57 509 Figure legends 58 510 59 60 61 62 63 64 65 511 Fig 1: The location of Lagoon and Anchorage Islands in Ryder Bay, south-eastern Adelaide 1 2 512 Island, off the west coast of the Antarctic Peninsula. 3 4 5 513 6 7 514 Figure 2. Maximum likelihood phylogenetic tree based on 16S rRNA gene sequences (1399 8 9 10 515 nt) of Pseudomonas versuta sp. nov. and the type strains of closely related Pseudomonas 11 12 516 species. Bootstrap values (only values > 50% expressed as percentages of 1000 replications) 13 14 517 are shown at the branching points. Bar, 5 nt substitutions per 100 nt. 15 16 17 518 18 19 519 Figure 3. Maximum Likelihood phylogenetic tree based on concatenated partial rpoD, rpoB 20 21 22 520 and gyrB gene sequences (2338 nt) of Pseudomonas versuta and the type strains of the 23 24 521 closely related Pseudomonas species. Bootstrap values (only values > 50% expressed as 25 26 27 522 percentages of 1000 replications) are shown at the branching points. Bar, 5 nt substitutions 28 29 523 per 100 nt. 30 31 32 524 33 34 525 35 36 526 37 38 39 527 40 41 528 42 43 44 529 45 46 530 47 48 49 531 50 51 532 52 53 533 54 55 56 57 58 59 60 61 62 63 64 65 Figure 1 Figure2 R1 Pseudomonas poae DSM 14936 T (AJ492829) 73 Pseudomonas trivialis DSM 14937 T (AJ492831) Pseudomonas costantinii CFBP 5705 T (AF374472) 56 84 Pseudomonas lurida DSM 15835 T (AJ581999) Pseudomonas antarctica CMS 35 T (AJ537601) 9494 Pseudomonas meridiana CMS 38 T (AJ537602) Pseudomonas extremaustralis CT14-3 T (AJ583501 ) Pseudomonas grimontii LMG 2123 T (AF268029) 81 Pseudomonas rhodesiae CIP 104664 T (AF064459) Pseudomonas migulae CIP 105470 T (AF074383)
T 52 Pseudomonas brenneri CFML 97-391 (AF268968) 81 Pseudomonas yamanorum 8H1 T (EU557337) Pseudomonas arsenicoxydans VC-1 T (FN645213) Pseudomonas prosekii AN/28/1 T (JN814372) Pseudomonas lini CFBP 5737 T (AY035996) Pseudomonas mediterranea CFBP 5447 T (AF386080) Pseudomonas thivervalensis CFBP 11261 T (AF100323) 63 Pseudomonas brassicacearum subsp. brassicacearum DBK11 T (AF100321) 54 Pseudomonas kilonensis 520-20 T (AJ292426) 73 Pseudomonas corrugata ATCC 29736 T (D84012) Pseudomonas chlororaphis subsp. aurantiaca NCIB 10068 T (DQ682655) Pseudomonas endophytica BSTT44 T (LN624760)
67 Pseudomonas psychrophila E3 T(AB041885) Pseudomonas fragi ATCC 4973 T (AF094733) Pseudomonas helleri DSM 29165 T (KP738715) 62 94 Pseudomonas lundensis ATCC 49968 T (AB021395) Pseudomonas taetrolens IAM 1653 T (D84027) Pseudomonas versuta L10.10 T (KX714290) 99 Pseudomonas deceptionensis M1 T (GU936597) Pseudomonas weihenstephanensis DSM 29166 T (KP738720) Pseudomonas asturiensis LPPA 221 T (FM865870) Pseudomonas amygdali LMG 2123 T (Z76654)
T 92 Pseudomonas ficuserectae JCM 2400 (AB021378) 88 Pseudomonas tremae CFBP 6111 T (AJ492826) Pseudomonas aeruginosa LMG 1242 T (Z76651) Permianibacter aggregans HW001 T (KJ721800)
0.050 Figure 3 77 Pseudomonas weihenstephanensis (JYLF00000000) Pseudomonas fragi ( FN554466, AJ717444, FN554184) Pseudomonas deceptionensis (GU936596, HE800510, HE800476) 72 80 Pseudomonas psychrophila (FN554506, AJ717464, FN554221) Pseudomonas versuta A4R1.12 ( KY264779, KY264780, KY264781 ) 62 100 Pseudomonas versuta A4R1.5 ( KY264776, KY264777, KY264778 ) 85 Pseudomonas versuta L10.10 T(KY264773, KY264774, KY264775 ) 99 Pseudomonas lundensis (FN554479, AJ717428, FN554197) 68 Pseudomonas taetrolens (AB039523, AJ717423, AB039412) Pseudomonas helleri (JYLD00000000) 100 Pseudomonas endophytica (LN624763, LN624764, LN624765) 80 100 Pseudomonas brenneri ( FN554457, AJ717482, FN554176) Pseudomonas meridiana ( FN554485, FN554740, FN554203) Pseudomonas antarctica (FN554450, FN554727, FN554169 Pseudomonas costantinii (FN554461, FN554732, FN554180) 97 Pseudomonas poae (FN554504, FN554751, FN554219) 79 Pseudomonas lurida (HE800497, HE586402, HE800480)
98 88 Pseudomonas extremaustralis (HE800495, JN814371, HE800479) Pseudomonas veronii LMG 17761(T)(FN554518, AJ717445, FN554233) Pseudomonas grimontii (FN554470, AJ717439, FN554188) 56 100 Pseudomonas panacis (FN554498, FN554748, FN554214) Pseudomonas arsenicoxydans (HE800488, HE800503, HE800469) 99 Pseudomonas migulae (FN554486, AJ717446, FN554204) Pseudomonas brassicacearum (AM084334 AJ717436 AM084675) 96 Pseudomonas kilonensis (AM084336, AJ717472, AM084677) Pseudomonas chlororaphis subsp. aureofaciens (FN554453, AJ717426, FN554172) Pseudomonas aeruginosa (AJ633568, AJ717442, AJ633104)
0.05 Table 1 R1
Table 1. Cellular fatty acid composition (%) of P. versuta L10.10 T, its phylogenetically closest related species and the type species of this genus, P. aeruginosa . Taxa: 1, P. versuta L10.10 T; 2, P. weihenstephanensis DSM 29166 T; 3, P. deceptionensis LMG25555 T; 4, P. endophytica BSTT44 T; 5, P. psychrophila LMG24276 T; 6, P. fragi LMG2191 T; 7, P. helleri DSM 29165 T; 8, P. taetrolens DSM 21104 T; 9, P. lundensis LMG13517 T; 10, P. aeruginosa ATCC 10145 T. Data were obtained in this study and from ∫Xiao et al. [56 ] using the same conditions. nd: no detected, tr: traces.
Fatty acids 1 2 3 4 5 6 7 8 9 ∫10 12:0 3.0 3.4 3.3 3.6 3.9 3.9 3.6 3.5 3.1 4.8 14:0 0.5 0.7 1.1 0.4 0.6 0.5 0.5 0.1 0.5 1.3 16:0 29.4 28.0 36.0 29.4 29.7 25.1 28.8 29.2 31.1 20.5 17:0 nd 0.2 0.1 0.1 0.1 0.2 0.2 0.2 0.2 tr 18:0 0.5 0.5 0.5 0.7 0.7 1.4 0.4 0.1 0.6 tr
10:0 3OH 4.6 5.3 4.0 3.6 4.1 4.4 7.2 3.1 3.9 3.6 12:0 2OH 5.2 5.1 4.0 3.4 3.4 3.7 6.3 3.4 4.1 3.7 12:0 3OH 5.6 5.4 4.5 4.2 4.3 4.9 6.7 5.1 3.9 4.5
17:0 cyclo 3.4 4.6 3.9 4.5 1.3 0.7 13.9 11.8 2.6 nd 19:0 cyclo 8c 0.5 0.8 0.1 0.2 nd 0.3 2.5 2.6 0.4 nd
Summed feature 3* 35.8 33.2 33.0 31.8 40.2 34.0 21.4 28.0 34.8 20.0 Summed feature 8¥ 11.5 12.6 8.1 16.5 11.3 19.1 8.0 12.5 13.6 38.9 *Summed feature 3: C16:1 7c/ C16:1 6c. ¥Summed feature 8: C18:1 7c / C18:1 6c . Table 2 R1
1 Table 2. Differential characteristics of P. versuta , its phylogenetically closest related species and the type species of this genus, P. aeruginosa . 2 Taxa: 1, P. versuta L10.10 T; 2, P. versuta A4R1.5; 3, P. versuta A4R1.12; 4, P. weihenstephanensis DSM 29166 T; 5, P. deceptionensis 3 LMG25555 T; 6, P. endophytica BSTT44 T; 7, P. psychrophila LMG24276 T; 8, P. fragi LMG2191 T; 9, P. helleri DSM 29165 T; 10, P. taetrolens 4 DSM 21104 T; 11, P. lundensis LMG13517 T; 12, P. aeruginosa ATCC 10145 T. Data were obtained in this study unless specifically indicated. +, 5 positive; -, negative; w, weak; nd, no data. 6 1 2 3 4 5 6 7 8 9 10 11 12¥ Fluorescent pigments King B Agar - - - + - + + + + - + + Growth at: 33 °C - - - + + - + + - + - + Production of: Arginine dihydrolase + + + + + + + + - w + + Assimilation of: D-Mannose + + + - + - + - w + - - D-Mannitol + + + - + - + - + + - + Phenylacetate ------+ - - - m-Inositol + + + + + - - - - + - - D-Trehalose + + + + + - w + + + - - Mono -methyl -succinate + - w + + - + + - - + + Acetate + + + + + - + + + + + + D-Galactonate acid lactone + + + - - + - - + + - - D-Galacturonate + + + + - + + - + + - + Itaconate ------+ + - + Succinam ate + - - + + + + + + - - + Glucuronamide + + + + + + + - + + - - L-Alaninamide + - + ------w - - L-Alanyl -glycine + + + + + - w + - - + nd Hydroxy -L-Proline + - - - + - + + - + - + D-L Carnitine + + + + - - - - w + - w Urocanate + + + + - - + + + + + - Inosine + + + + + - + - - + - + Uridine + + + - - - w - - + - w
Genome feature: Genome size (Mb) 5.15 5.20 5.10 4.79 5.05 4.97 5.33 5.02 5.67 4.92 4.99 6.29 DNA G+C content (mol %) 58.2 58.2 58.3 57.3 58.6 55.2 57.5 59.4 58.1 58.3 58.5 66.6 Number of genes # 4639 4774 4690 4417 4631 4598 4889 4557 5040 4481 4609 5787 Number of coding sequences # 4425 4636 4553 4196 4460 4276 4718 4297 4811 4300 4365 5698 ¥ 7 Data for P. aeruginosa ATCC 10145 T are from Palleroni [34], Clark et al. [9] and Xiao et al. [56] Supplementary Material Click here to download Supplementary Material: Fig. S1R1.pptx Figure S2 Click here to download Supplementary Material: Fig. S2.pptx Figure S3 Click here to download Supplementary Material: Fig. S3.pptx Table S1 Click here to download Supplementary Material: Table S1.docx Table S2 Click here to download Supplementary Material: Table S2.docx digital protologue pdf Click here to download Supplementary Material: dprotologue.pdf culture collection deposit certificate Click here to download Supplementary Material: Pseudomonas strain L1010 letter LMG 29628.pdf culture collection deposit certificate Click here to download Supplementary Material: Pseudomonas strain L1010 letter of deposition DSM 101070.pdf