*Manuscript 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.