Pseudomonas guineae sp. nov., a novel psychrotolerant bacterium from an Antarctic environment Nu ´ ria Bozal, M. Jesu ´ s Montes and Elena Mercade ´ Correspondence Elena Mercade ´ mmercade@ub.edu Laboratori de Microbiologia, Facultat de Farmacia, Universitat de Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain Two Gram-negative, cold-adapted, aerobic bacteria, designated strains M8 T and M6, were isolated from soil collected from the South Shetland Islands. The organisms were rod-shaped, catalase- and oxidase-positive and motile by means of polar flagella. These two psychrotolerant strains grew between ”4 and 30 6C. 16S rRNA gene sequence analysis placed strains M8 T and M6 within the genus Pseudomonas. DNA–DNA hybridization experiments between the Antarctic isolate M8 T and type strains of phylogenetically related species, namely Pseudomonas peli and Pseudomonas anguilliseptica, revealed levels of relatedness of 33 and 37 %, respectively. Strain M6 showed 99 % DNA similarity to strain M8 T . Several phenotypic characteristics, together with data on cellular fatty acid composition, served to differentiate strains M8 T and M6 from related pseudomonads. On the basis of the polyphasic taxonomic evidence presented in this study, it can be concluded that strains M8 T and M6 belong to the same genospecies, representing a novel species of the genus Pseudomonas, for which the name Pseudomonas guineae sp. nov. is proposed. The type strain is M8 T (5LMG 24016 T 5CECT 7231 T ). In recent years, increasing attention has been devoted to cold-adapted micro-organisms and their enzymes (Antranikian et al., 2005). Antarctica has become a great source of novel psychrophilic and psychrotolerant strains, some of which belong to the genus Pseudomonas (Kriss et al., 1976; Shivaji et al., 1989; Ma et al., 2006; Maugeri et al., 1996; Bruni et al., 1999; Reddy et al., 2004). The genus Pseudomonas once comprised more than 100 species, but, over the last decade, many of these have been reclassified into different genera (Kersters et al., 1996; Anzai et al., 2000). During a taxonomic investigation of cold-adapted bacteria from soil samples collected in the Antarctic area of the South Shetland Islands, two strains, M6 and M8 T , able to grow at 24 uC and capable of forming swarming colonies on trypticase soy agar (TSA) were isolated. In this study, the taxonomic status of these two strains was investigated by using a combination of phenotypic characterization, 16S rRNA gene sequencing, DNA G+C content determination, DNA–DNA hybridiza- tion and cellular fatty acid analysis. The data obtained show that strains M6 and M8 T belong to a novel species of the genus Pseudomonas, for which the name Pseudomonas guineae sp. nov. is proposed, with M8 T as the type strain. Strains M8 T and M6 were isolated from a soil sample collected from Deception Island (South Shetland Islands, Antarctica). Sample aliquots were removed with a platinum loop and diluted in a saline solution (pH 7) containing the following salts (g l 21 ): NaCl, 0.56; KCl, 0.027; CaCl 2 , 0.03; NaHCO 3 , 0.01. TSA plates were inoculated with loopfuls of several sample dilutions by using the streak-plate method to obtain isolated colonies. Plates were incubated for 4 days at 15 u C. Isolates were maintained aerobically on TSA slopes at 4 u C and also at 280 uC on cryo-beads. The morphology, cell size and shape of cells grown on TSA at 15 u C were determined by means of negative staining and transmission electron microscopy. Motility was determined by phase-contrast microscopy. Oxidase, cata- lase and urease activities, nitrate reduction and hydrolysis of casein, lecithin, gelatin, DNA, starch and Tween 80 were determined according to Cowan & Steel (1993). The presence of fluorescent pigments was tested under UV light after 8 days on King’s B medium (King et al., 1954). Acid production from carbohydrates, enzyme production and additional characteristics were determined by using API 50 CH, API ZYM and API 20NE strips (bioMe ´rieux). Tolerance of NaCl was measured on nutrient agar The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains M8 T and M6 are AM491810 and AM491811, respectively. An electron micrograph of a cell of strain M8 T , an extended phylogenetic tree for strains M8 T and M6 within the genus Pseudomonas and a table showing cellular fatty acid compositions are available with the online version of this paper. International Journal of Systematic and Evolutionary Microbiology (2007), 57, 2609–2612 DOI 10.1099/ijs.0.65141-0 65141 G 2007 IUMS Printed in Great Britain 2609