Systematic and Applied Microbiology 37 (2014) 89–94 Contents lists available at ScienceDirect Systematic and Applied Microbiology j ourna l h omepage: www.elsevier.de/syapm Pseudomonas aestusnigri sp. nov., isolated from crude oil-contaminated intertidal sand samples after the Prestige oil spill  David Sánchez a , Magdalena Mulet a , Ana C. Rodríguez a , Zoyla David a , Jorge Lalucat a,b , Elena García-Valdés a,b,∗ a Microbiologia, Departament de Biologia, Edifici Guillem Colom, Universitat de les Illes Balears, Campus UIB, 07122 Palma de Mallorca, Spain b Institut Mediterrani d’Estudis Avanc ¸ ats (IMEDEA, CSIC-UIB), Campus UIB, 07122 Palma de Mallorca, Spain a r t i c l e i n f o Article history: Received 11 June 2013 Received in revised form 5 September 2013 Accepted 6 September 2013 Keywords: Pseudomonas sp. nov. Bacteria Taxonomy a b s t r a c t Strains VGXO14 T and Vi1 were isolated from the Atlantic intertidal shore from Galicia, Spain, after the Prestige oil spill. Both strains were Gram-negative rod-shaped bacteria with one polar inserted flagel- lum, strictly aerobic, and able to grow at 18–37 ◦ C, pH 6–10 and 2–10% NaCl. A preliminary analysis of the 16S rRNA and the partial rpoD gene sequences indicated that these strains belonged to the Pseudomonas genus but were distinct from any known Pseudomonas species. A polyphasic taxonomic approach includ- ing phylogenetic, chemotaxonomic, phenotypic and genotypic data confirmed that the strains belonged to the Pseudomonas pertucinogena group. In a multilocus sequence analysis, the similarity of VGXO14 T and Vi1 to the closest type strain of the group, Pseudomonas pachastrellae, was 90.4%, which was lower than the threshold of 97% established to discriminate species in the Pseudomonas genus. The DNA–DNA hybridisation similarity between strains VGXO14 T and Vi1 was 79.6%, but below 70% with the type strains in the P. pertucinogena group. Therefore, the strains should be classified within the genus Pseudomonas as a novel species, for which the name Pseudomonas aestusnigri is proposed. The type strain is VGXO14 T (=CCUG 64165 T = CECT 8317 T ). © 2013 Elsevier GmbH. All rights reserved. Introduction The bacteria of the genus Pseudomonas are Gram-negative, strictly oxidative rods within the Gammaproteobacteria that are motile by polar flagella [17]. The genus Pseudomonas is ubiquitous, metabolically versatile, and important for the recycling of elements in the environment. Pseudomonas is the genus of Gram-negative bacteria with the highest number of species, and new species are described continuously. In 2012, two novel species were added to the genus: P. nitritireducens [22] and P. sagittaria [9]. At the time of this manuscript’s composition, 139 validated species were listed in Euzeby’ list of prokaryotic names with standing in nomenclature (2013) (http://www.bacterio.cict.fr/). The characterisation of Pseudomonas-type strains by multilo- cus sequence analysis (MLSA), and concatenating the 16S rDNA,  The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences reported in this study are: HG004394 (strain P. aestusnigri VGXO14 T 16S rRNA), HG004395 (strain P. aestusnigri Vi1 16S rRNA), HG004396 (strain P. aestusnigri VGXO14T gyrB gene) and HG004397 (strain P. aestusnigri Vi1 gyrB gene). ∗ Corresponding author at: Microbiologia, Departament de Biologia, Edifici Guillem Colom, Universitat de les Illes Balears, Crtra. de Valldemosa Km 7,5, 07122 Palma de Mallorca, Spain. Tel.: +34 971 173141; fax: +34 971 173184. E-mail address: elena.garciavaldes@uib.es (E. García-Valdés). gyrB, rpoD and rpoB genes permitted the establishment of two main lineages, P. fluorescens and P. aeruginosa, which were divided into several groups (G) and subgroups (SG). Some type strains did not belong to any of these lineages, since they were located in an independent phylogenetic branch, including those representing the species P. oryzihabitans, P. psychrotolerans, P. luteola, P. pertu- cinogena and P. pachastrellae [14]. In 2012, five type strains were analysed by MLSA and they were shown to be phylogenetically close to P. pertucinogena. The strains are now considered to be members of a new group, the P. pertucinogena group [15], which is represented by type strains P. bauzanensis DSM 22558 T [23], P. litoralis CECT 7670 T [18], P. pelagia JCM 15562 T [2], P. sabulinigri KCTC 22137 T [5] and P. xiamenensis JCM 13530 T [7]. A previous study [13] on the diversity of autochthonous Pseu- domonas populations in two contaminated beaches after the Prestige oil spill (Carnota municipality, Galicia, north-western Spain) in November 2002, rendered 102 Pseudomonas isolates from intertidal sand contaminated with crude oil. From this analysis, several isolates were considered as possible new species. These results were confirmed by the analysis of partial sequences of the sigma 70 subunit of RNA polymerase, encoded by the rpoD gene. A deep taxonomic study of the characteristics of two of these strains is presented. A new Pseudomonas species in the P. pertucinogena group, with isolate VGXO14 T as the type strain, is proposed. Strain 0723-2020/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.syapm.2013.09.004