International Journal of Systematic and Evolutionary Microbiology (2014), 64, 2338–2345 DOI 10.1099/ijs.0.063560-0 Pseudomonas helmanticensis sp. nov., isolated from forest soil Martha-Helena Ramı´rez-Bahena,1,2 Maria Jose´ Cuesta,1 Jose´ David Flores-Fe´lix,3 Rebeca Mulas,4 Rau´l Rivas,2,3 Joao Castro-Pinto,5 Javier Bran˜as,5 Daniel Mulas,5 Fernando Gonza´lez-Andre´s,4 Encarna Vela´zquez2,3 and A´ lvaro Peix1,2 Correspondence 1Instituto de Recursos Naturales y Agrobiologı´a, IRNASA-CSIC, Salamanca, Spain A´ lvaro Peix 2Unidad Asociada Grupo de Interaccio´n Planta-Microorganismo, [email protected] Universidad de Salamanca-IRNASA (CSIC), Salamanca, Spain 3Departamento de Microbiologı´a y Gene´tica, Universidad de Salamanca, Salamanca, Spain 4Instituto de Medio Ambiente, Recursos Naturales y Biodiversidad, Universidad de Leo´n, Leo´n, Spain 5Fertiberia S. A., Madrid, Spain A bacterial strain, OHA11T, was isolated during the course of a study of phosphate-solubilizing bacteria occurring in a forest soil from Salamanca, Spain. The 16S rRNA gene sequence of strain OHA11T shared 99.1 % similarity with respect to Pseudomonas baetica a390T, and 98.9 % similarity with the type strains of Pseudomonas jessenii, Pseudomonas moorei, Pseudomonas umsongensis, Pseudomonas mohnii and Pseudomonas koreensis. The analysis of housekeeping genes rpoB, rpoD and gyrB confirmed its phylogenetic affiliation to the genus Pseudomonas and showed similarities lower than 95 % in almost all cases with respect to the above species. Cells possessed two polar flagella. The respiratory quinone was Q9. The major fatty acids were C16 : 0, C18 : 1v7c and summed feature 3 (C16 : 1v7c/iso-C15 : 0 2-OH). The strain was oxidase-, catalase- and urease-positive, positive for arginine dihydrolase but negative for nitrate reduction, b-galactosidase production and aesculin hydrolysis. It was able to grow at 31 6C and at pH 11. The DNA G+C content was 58.1 mol%. DNA–DNA hybridization results showed values lower than 49 % relatedness with respect to the type strains of the seven closest related species. Therefore, the combined genotypic, phenotypic and chemotaxonomic data support the classification of strain OHA11T to a novel species of the genus Pseudomonas, for which the name Pseudomonas helmanticensis sp. nov. is proposed. The type strain is OHA11T (5LMG 28168T5CECT 8548T). Many species of bacteria are able to solubilize inorganic the recently described Pseudomonas guariconensis (Toro et al., phosphates in vitro, and some of them can mobilize 2013). During a study of phosphate-solubilizing rhizospheric phosphorus to plants (Antoun et al., 1998; Peix et al., 2001; bacteria in soils from northern Spain, we isolated a strain Kaur & Reddy, 2013). Within soil microbiota, the groups (OHA11T) in a forest soil located in Salamanca province that of pseudomonads, bacilli and rhizobia are considered the is able to produce a large transparent ‘halo’ surrounding its most effective phosphate solubilizers (Rodrı´guez & Fraga, colonies in media containing insoluble bicalcium phosphate 1999). The genus Pseudomonas includes several species as the phosphorus source, and subjected this strain to a reported as phosphate-solubilizing bacteria, some of which polyphasic taxonomic study. The isolation procedure was the were isolated from soil, such as Pseudomonas rhizosphaerae same as described by Peix et al. (2003).Briefly,10gof (Peix et al., 2003), Pseudomonas lutea (Peix et al., 2004) or rhizospheric soil was suspended in 90 ml sterile water and stirred for 30 min. From this suspension, 100 mlwasspread on YED-P medium and incubated at 28 uC for 7 days. The The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, isolate formed a transparent ‘halo’ around the colonies, rpoD rpoB gyrB T , and gene sequences of strain OHA11 are HG940537, indicating in vitro phosphate solubilization activity. HG940517, HG940518 and HG940516, respectively. One supplementary table and one supplementary figure are available This strain was classified to the genus Pseudomonas within with the online version of this paper. the Gammaproteobacteria based on 16S rRNA and Downloaded from www.microbiologyresearch.org by 2338 063560 G 2014 IUMS Printed in Great Britain IP: 193.146.109.16 On: Wed, 06 Jul 2016 18:52:00 Pseudomonas helmanticensis sp. nov. housekeeping gene sequence analyses, and the phyloge- including all the closest related species to the novel strain as netic, chemotaxonomic and phenotypic data obtained well as the type species of the genus, Pseudomonas aeruginosa showed that it represents a novel species. LMG 1242T. According to the maximum-likelihood phylo- genetic tree (Fig. 1), strain OHA11T clustered in a separate Cells were stained according to the Gram procedure branch related to P. baetica a390T. The results were described by Doetsch (1981). Motility was checked by congruent with the tree topology obtained after neigh- phase-contrast microscopy after growing cells in nutrient bour-joining phylogenetic analysis (data not shown). agar medium at 22 uC for 48 h. The flagellation type was determined by electron microscopy after 48 h of incuba- Additionally to the 16S rRNA gene, three housekeeping tion on tripticase soy agar (TSA; Becton Dickinson, BBL) at genes widely used in the phylogenetic analysis of species of 22 uC as previously described (Rivas et al., 2007). Strain the genus Pseudomonas were analysed here (Tayeb et al., OHA11T was Gram-stain-negative, rod-shaped (0.6–0.86 2005; Mulet et al., 2009, 2010, 2012; Ramos et al., 2013; 1.6–2.0 mm) and motile by means of two polar flagella (Fig. Toro et al., 2013). The phylogenies obtained for the three S1, available in the online Supplementary Material). Cells genes were congruent with those based on the 16S rRNA grew as round translucent beige to yellowish colonies on gene sequence analysis, supporting the affiliation of strain nutrient agar. For 16S rRNA gene sequencing and compar- OHA11T to the genus Pseudomonas as a separate species ison analysis, DNA extraction, amplification and sequencing related to the P. baetica cluster. The concatenated rpoD, were performed as reported by Rivas et al. (2007). Ampli- rpoB and gyrB gene phylogenetic tree showed that strain T fication and partial sequencing of the gyrB, rpoB and rpoD OHA11 clusters in a separate branch that is related to a housekeeping genes were performed as described by Mulet group formed by P. baetica, P. koreensis and Pseudomonas et al. (2010), using the primers PsEG30F (59-ATYGAA- moraviensis (Fig. 2). Levels of rpoD, rpoB and gyrB gene ATCGCCAARCG-39) and PsEG790R (59-CGGTTGATKT- sequence similarity were about 87–92, 94–95 and 93–97 %, T CCTTGA-39) for the rpoD gene (Mulet et al., 2009), LAPS5F respectively, between strain OHA11 and the type strains of (59-TGGCCGAGAACCAGTTCCGCGT-39) and LAPS27R P. baetica, P. jessenii, P. moorei, P. mohnii, P. umsongensis, (59-CGGCTTCGTCCAGCTTGTTCAG-39) for the rpoB gene P. koreensis and P. reinekei. These values are similar to or (Tayeb et al., 2005), and GyrBPUN1F (59-AAGGAGCTGG- lower than those found among several species of the genus TGYTGACC-39) and GyrBPUN1R (59-GCGTCGATCAT- Pseudomonas. For example, in the case of the rpoD gene, CTTGCCG-39) for the gyrB gene (Ramos et al., 2013). the type strain of P. jessenii showed about 92 % similarity with respect to those of Pseudomonas vancouverensis, P. The sequences obtained (1472 nt for 16S, 721 nt for rpoD, moorei and P. mohnii; the type strain of P. reinekei showed 1125 nt for rpoB and 713 nt for gyrB) were compared with 94 % similarity with respect to those of P. moorei and P. those from GenBank using the BLASTN (Altschul et al., mohnii; the type strains of P. moorei and P. mohnii showed 1990) and EzTaxon (Kim et al., 2012) programs and levels 96 % similarity between themselves and 93.7 % similarity of similarity were calculated after pairwise comparison. For with those of P. koreensis and P. moraviensis; and the type phylogenetic analysis, sequences were aligned using the strain of Pseudomonas punonensis showed 91.6 % similarity CLUSTAL X software (Thompson et al., 1997). Distances were with respect to those of Pseudomonas argentinensis and calculated according to Kimura’s two-parameter model Pseudomonas straminea. In the case of the rpoB gene, the (Kimura, 1980). Phylogenetic trees based on 16S rRNA type strains of P. vancouverensis and P. mohnii shared gene sequences were reconstructed using the neighbour- 95.6 % similarity; the type strains of P. moorei and P. joining (Saitou & Nei, 1987) and maximum-likelihood mohnii, P. jessenii and P. reinekei, P. koreensis and P. (Rogers & Swofford, 1998) methods. The MEGA5 software moraviensis, and P. vancouverensis, P. jessenii and P. reinekei (Tamura et al., 2011) was used for all analyses. shared about 97 % identity; the type strain of P. punonensis Comparison of the 16S rRNA gene sequence of strain had 95.8 % similarity with respect to that of P. argentinensis OHA11T against the type strains of bacterial species and 90.5–90.7 % with respect to those of P. straminea and recorded in the EzTaxon-e database showed the affiliation Pseudomonas flavescens; and the type strain of P. of the novel strain to the genus Pseudomonas. The closest guariconensis had 86 % similarity with respect to that of relative was Pseudomonas baetica a390T (Lo´pez et al., 2012) Pseudomonas entomophila and 84–85 % with respect to with 99.1 % pairwise similarity (13 nt difference), and the those of Pseudomonas plecoglossicida, Pseudomonas monteilii next closest relatives with 98.9 % similarity (15–17 nt and Pseudomonas taiwanensis. All these species showed gyrB differences) were the type strains of Pseudomonas jessenii gene sequence similarities ranging from 86 to 97 %. Therefore, the results of the rpoD, rpoB and gyrB gene (Verhille et al., 1999), Pseudomonas moorei, Pseudomonas sequence analyses also indicated that strain OHA11T mohnii (Ca´mara et al., 2007), Pseudomonas umsongensis represents an undescribed species of the genus Pseudomonas.