Pseudomonas pachastrellae sp. nov., isolated from a marine sponge

Two Gram-negative, non-fermentative, non-denitrifying, non-pigmented, rod-shaped bacteria that were motile by means of polar flagella, designated strains KMM 330^T and KMM 331, were isolated from a deep-sea sponge specimen and subjected to a polyphasic taxonomic study. The new isolates exhibited 16S rRNA gene sequence similarity of 99·9 %, and their mean level of DNADNA relatedness was 82 %. Phylogenetic analysis based on their 16S rRNA gene sequences placed the strains within the genus Pseudomonas as an independent deep clade. Strain KMM 330^T shared highest sequence similarity (96·3 %) with each of Pseudomonas fulva NRIC 0180^T, Pseudomonas parafulva AJ 2129^T and Pseudomonas luteola IAM 13000^T; sequence similarity to other recognized species of the genus Pseudomonas was below 95·7 %. The marine sponge isolates KMM 330^T and KMM 331 could be distinguished from the other recognized Pseudomonas species based on a unique combination of their phenotypic characteristics, including growth in 8 or 10 % NaCl, the absence of pigments, the inability to denitrify and lack of carbohydrate utilization. On the basis of phylogenetic analysis, physiological and biochemical characterization, strains KMM 330^T and KMM 331 should be classified as a novel species of the genus Pseudomonas, for which the name Pseudomonas pachastrellae sp. nov. is proposed. The type strain is KMM 330^T (=JCM 12285^T=NRIC 0583^T=CCUG 46540^T).

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Abbreviations: DPG, diphosphatidylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol

Published online ahead of print on 18 November 2004 as DOI 10.1099/ijs.0.63176-0.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of KMM 330^T and KMM 331 are AB125366 and AB125367, respectively.

Images of cell morphology, a thin-layer chromatogram of polar lipids and a maximum-likelihood tree are available as supplementary figures in IJSEM Online.

Pseudomonads are well-known and widespread micro-organisms, which have been isolated from a variety of natural sources, soil, plants, mineral waters and clinical specimens, and they are characterized by a high level of metabolic diversity (Rosselló et al., 1991; Moore et al., 1996). The vast majority of species formerly assigned to Pseudomonas and recovered from marine sources are now classified within other genera (Kersters et al., 1996; Brown et al., 2001; Yoon et al., 2003). Relatively few species originating from marine environments are included within Pseudomonas (sensu stricto), including Pseudomonas stutzeri (later synonym Pseudomonas perfectomarina) (Baumann et al., 1983; Rosselló-Mora et al., 1993), Pseudomonas alcaligenes, Pseudomonas pseudoalcaligenes (Palleroni, 1984) and Pseudomonas alcaliphila (Yumoto et al., 2001) from sea waters, and some strains of Pseudomonas balearica (Bennasar et al., 1996) have been recovered from marine sediments.

In the present study two Pseudomonas-like bacteria isolated from a deep-sea sponge specimen were investigated by using a polyphasic taxonomic approach. The new strains KMM 330^T and KMM 331 appeared to have homogeneous phenotypic characteristics and to be closely related based on phylogenetic and DNADNA hybridization experiments, but clearly distinct from all recognized Pseudomonas species. Based on the phenotypic and molecular data obtained, a novel species, Pseudomonas pachastrellae sp. nov., is described.

Two strains, KMM 330^T and KMM 331, were isolated from a sponge specimen of Pachastrella sp., collected in 1991 from the Philippine Sea at a water depth of 750 m. A small amount of internal tissue of the sponge was homogenized and aliquots of the diluted homogenates were spread on agar plates of sea water medium (SWM), containing: 5·0 g peptone l¹, 2·5 g yeast extract l¹, 1·0 g glucose l¹, 0·2 g K₂HPO₄ l¹, 0·05 g MgSO₄ l¹ and 15·0 g agar l¹ in 750 ml sea water/250 ml distilled water. The inoculated plates were incubated for 14 days at 28 °C. Each colony was picked, re-streaked on the agar nutrient media and tested. The bacteria were grown aerobically on marine 2216 agar (MA) or marine broth (MB; Difco) and trypticase soy agar (TSA) at 2528 °C. They were also stored at 80 °C in the liquid medium supplemented with 30 % (v/v) glycerol. Strains KMM 330^T and KMM 331 have been deposited in the Collection of Marine Micro-organisms (KMM) of the Pacific Institute of Bioorganic Chemistry, Vladivostok, Russia. The following Pseudomonas type strains, P. stutzeri CIP 103022^T, P. alcaligenes CIP 101034^T, P. pseudoalcaligenes CIP 66.14^T and Pseudomonas mendocina CIP 75.21^T, were kindly provided by the Collection de l'Institut Pasteur, Paris, France. Motility was observed by the hanging drop method. Cell morphology was examined by transmission electron microscopy from exponential phase cells grown in MB. Cells were negatively stained with potassium phosphotungstate (1 %, w/v; pH 7·0). Production of pyocyanin and formation of fluorescent pigments were tested on King A and King B medium, respectively (King et al., 1954). The Gram-reaction, oxidase and catalase reactions and production of amylase, caseinase, DNase, gelatinase and lipase (Tween 80) were determined according to the methods described by Smibert & Krieg (1994). Acid production from carbohydrates was examined by using the medium of Leifson (1963) for marine bacteria. The API 20NE, API 32GN and API ZYM systems (bioMérieux) were additionally used to test biochemical properties and these tests were performed according to the manufacturer's instructions. The temperature range for growth was determined on TSA and MA at 4, 7, 12, 28, 37, 41, 42 and 43 °C. The pH range for growth (5·010·0) was tested using trypticase soy broth (TSB) and MB. Growth requirement for sodium ions and salt tolerance were examined on SWM prepared on a distilled water base supplemented with an appropriate amount of NaCl ranging from 0 to 15 % (w/v). DNA was isolated by the procedure of Marmur (1961). DNA base composition was determined as described by Marmur & Doty (1962) and Owen et al. (1969). The level of DNADNA relatedness between strains KMM 330^T and KMM 331 was measured spectrophotometrically by using the initial renaturation rate method of De Ley et al. (1970). The photobiotin-labelled DNA probe microplate method of Ezaki et al. (1989) was used to determine genetic relatedness of strain KMM 330^T and selected type strains of Pseudomonas species, including Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas fulva, Pseudomonas luteola, Pseudomonas parafulva, Pseudomonas putida, Pseudomonas straminea and P. stutzeri. Bacteria were grown on TSA at 28 °C for 48 h and whole-cell fatty acids were determined in accordance with the procedure described by Svetashev et al. (1995). Cellular quinones were determined by HPLC (LC-6A; Shimadzu) using a Cosmosil 5C18 column (4·6x150 mm), with acetonitril/2-propanol (50 : 50, v/v) as the eluant (0·5 ml min¹), at a temperature 40 °C and with an SPD-2AM (270 nm) UV detector. Polar lipids were examined as described by Vaskovsky & Terekhova (1979). 16S rRNA gene sequences of the strains tested were determined and compared as described by Shida et al. (1997). The sequences obtained were compared with 16S rRNA gene sequences retrieved from the EMBL/GenBank/DDBJ databases by using the FASTA program (Pearson & Lipman, 1988). Distances were calculated according to the method of Jukes & Cantor (1969). Phylogenetic trees were constructed by the neighbour-joining method of Saitou & Nei (1987) with the CLUSTAL X program (version 1.8; Thompson et al., 1997) and by the maximum-likelihood method using the BIOEDIT program (Hall, 1999).

The marine isolates KMM 330^T and KMM 331 were Gram-negative, non-pigmented, rod-shaped encapsulated bacteria that were motile by means of a single polar flagellum (see Supplementary Fig. A in IJSEM Online). Physiological, biochemical and chemotaxonomic characteristics of strains KMM 330^T and KMM 331 are given in Table 1 and under the species description below.

Table 1. Phenotypic characteristics of Pseudomonas pachastrellae sp. nov. and other Pseudomonas species Strains/species: 1, P. pachastrellae sp. nov. strains KMM 330T and KMM 331; 2, P. aeruginosa; 3, P. fluorescens biovar 1; 4, P. putida biovars; 5, P. straminea; 6, P. luteola; 7, P. fulva; 8, P. parafulva; 9, P. stutzeri; 10, P. balearica; 11, P. mendocina; 12, P. alcaligenes; 13, P. pseudoalcaligenes; 14, P. alcaliphila. Data are from the present study and from Palleroni (1984), Uchino et al. (2000, 2001), Anzai et al. (1997), Bennasar et al. (1996) and Yumoto et al. (2001). +, Positive; , negative; d, different reaction between strains; ND, not determined.

The polar lipid compositions of strains KMM 330^T and KMM 331 were identical (see Supplementary Fig. B in IJSEM Online) and consistent in their major components with those previously published for other Pseudomonas species. The presence of phosphatidylethanolamine (PE) as the predominant phospholipid, with smaller amounts of phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG), in the fluorescent pseudomonads and their close relatives has been previously reported by Wilkinson (1988). Polar lipids consisting of PE, PG, DPG, phosphatidylcholine (PC), unknown phospholipids, unknown aminophospholipids and unknown polar lipids were detected in Pseudomonas oleovorans and Pseudomonas psychrotolerans by Hauser et al. (2004). The polar lipids of strains KMM 330^T and KMM 331 did not include PC or aminophospholipid components.

Strains KMM 330^T and KMM 331 contained isoprenoid quinone Q₉ and C_{16 : 0}, C_{16 : 1}ω9c and C_{18 : 1} as the dominant fatty acids, in agreement with data previously reported for Pseudomonas species (Oyaizu & Komagata, 1983) and for P. stutzeri strains (Rainey et al., 1994), P. fulva, P. parafulva and related species (Uchino et al., 2001) and P. alcaliphila (Yumoto et al., 2001).

The DNA G+C contents of the new isolates KMM 330^T and KMM 331 were 61·1 and 61·5 mol%, respectively, which is within the range reported for most Pseudomonas species (Palleroni, 1984).

On the basis of their quinone, polar lipid and fatty acid compositions, DNA base composition, cell morphology and motility and physiological characteristics, the isolates can be considered to be members of the genus Pseudomonas.

Strains KMM 330^T and KMM 331 had 16S rRNA gene sequence similarity of 99·9 %. P. fulva NRIC 0180^T, P. parafulva AJ 2129^T and P. luteola IAM 13000^T were the closest phylogenetic relatives (each 96·3 % 16S rRNA gene sequence similarity). Strains KMM 330^T and KMM 331 shared 95·7 % 16S rRNA gene sequence similarity with P. straminea IAM 1598^T and 95·3 % similarity with Pseudomonas denitrificans IAM 12023. These values fall within the range of 16S rRNA gene sequence similarities (93·799·9 %) reported for species of the genus Pseudomonas (Moore et al., 1996; Anzai et al., 2000). 16S rRNA gene sequence similarity values of below 97 % have been stated by Stackebrandt & Goebel (1994) to be an accepted criterion for differentiation of bacteria at the species level. Relatively low 16S rRNA gene sequence similarities (96·3 % to P. luteola IAM 13000^T, P. fulva NRIC 0180^T and P. parafulva AJ 2129^T, 95·7 % to P. straminea IAM 1598^T and values below this to other Pseudomonas species) clearly demonstrated that the new isolates could be regarded as members of an independent species. Phylogenetic analysis based on the 16S rRNA gene sequences revealed a clear affiliation of the new isolates to members of the genus Pseudomonas. Different algorithms placed strains KMM 330^T and KMM 331 as a separate clade adjacent to P. luteola IAM 13000^T and to the Pseudomonas pertucinogena IFO 14163^T and P. denitrificans IAM 12023 cluster (Fig. 1; see also Supplementary Fig. C in IJSEM Online). The species relatedness of the P. pertucinogena cluster and P. luteola among Pseudomonas species found in our analyses has been observed in other phylogenetic studies (Anzai et al., 1997, 2000; Peix et al., 2003).

(48K): Fig. 1. Phylogenetic relationships of isolates KMM 330T and KMM 331 and some related Pseudomonas species on the basis of 16S rRNA gene sequence analysis. The branching pattern was generated by the neighbour-joining method. Numbers indicate bootstrap values greater than 700. Bar, 0·01 Knuc units. A tree generated by the maximum-likelihood method is available as Supplementary Fig. C in IJSEM Online.

Strains KMM 330^T and KMM 331 possessed a high level of DNADNA relatedness (82 %), demonstrating their affiliation to the same species in accordance with the cut-off value of 70 % recognized by Wayne et al. (1987) for discrimination of bacterial species. Strain KMM 330^T exhibited low DNADNA relatedness with a range of closely and more distantly related type strains of P. aeruginosa (7 %), P. fluorescens (6 %), P. fulva (6 %), P. luteola (3 %), P. parafulva (7 %), P. putida (8 %), P. straminea (9 %) and P. stutzeri (9 %). Based on the 16S rRNA gene sequence similarity values (<97 %), their placement in the phylogenetic tree (Fig. 1) and levels of DNADNA relatedness, isolates KMM 330^T and KMM 331 should be classified as a novel species within the genus Pseudomonas.

The phylogenetic distinctiveness found for strains KMM 330^T and KMM 331 was supported by a combination of phenotypic features that does not allow their assignment to any recognized species of the genus Pseudomonas. The new marine isolates displayed a number of phenotypic characteristics that differentiate them from representatives of each of their related phylogenetic groups, including fluorescent, non-pigmented denitrifying or intracellular-pigmented Pseudomonas species. The phylogenetic relatives P. fulva, P. parafulva and P. luteola could be distinguished from strains KMM 330^T and KMM 331 based on the presence of intracellular pigments and utilization of various compounds. Strains KMM 330^T and KMM 331 were consistent with P. alcaligenes in their lack of carbohydrate utilization, but they differed in their inability to denitrify or to produce arginine dihydrolase and in their ability to grow in 810 % NaCl. In addition, the DNA G+C content of strains KMM 330^T and KMM 331 (61·161·5 mol%) clearly distinguished them from P. alcaligenes (6468 mol%) (Palleroni, 1984). Differential physiological and biochemical characteristics of the new marine isolates and a range of Pseudomonas species are summarized in Table 1. The most significant differential characteristics for strains KMM 330^T and KMM 331 are the inability to produce pigments, to denitrify, to produce arginine dihydrolase and to grow at 4 °C and the ability to grow in the presence of 810 % NaCl.

On the basis of combined phenotypic and phylogenetic data, the marine sponge isolates KMM 330^T and KMM 331 are considered to represent a novel species, for which the name Pseudomonas pachastrellae sp. nov. is proposed.

Description of Pseudomonas pachastrellae sp. nov.
Pseudomonas pachastrellae (pa.cha.strel'lae. N.L. gen. n. pachastrellae of Pachastrella, the generic name of a sponge).

Gram-negative, aerobic, non-pigmented, encapsulated, rod-shaped cells, 1·41·6 µm long and 0·40·5 µm in diameter, that are motile by a single flagellum. Oxidase- and catalase-positive. Forms circular, smooth, non-pigmented, whitish and transparent colonies, 23 mm in diameter, on TSA and MA. Pyocyanin on King A medium and fluorescent pigments on King B medium are not produced. A slightly yellowish diffusible pigment is observed during growth of strain KMM 330^T on King A medium. Growth is observed in 010 % (w/v) NaCl and at temperatures of 741 °C; no growth occurs at 4 or 42 °C. Acid is not produced from D-glucose, D-xylose, rhamnose, galactose, maltose, lactose or mannitol. Variable production of acid from glycerol. Hydrolysis of Tween 40 is positive. In addition to the physiological characteristics given in Table 1, the strains are negative for urease, lysine decarboxylase, ornithine decarboxylase, acetoin production, H₂S production, indole production, aesculin and DNA hydrolysis, haemolysis and utilization of L-arabinose, phenylacetate, DL-norleucine and N-acetylglucosamine and positive for citrate and DL-lactate utilization. In APY ZYM tests, strains are positive for esterase C4, esterase lipase C8, phosphoamidase, leucine arylamidase reaction and negative for alkaline phosphatase, lipase C14, valine arylamidase, cystine arylamidase, α-chymotrypsin, trypsin, acid phosphatase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. The major isoprenoid quinone is Q₉. The phospholipids are PE, PG, DPG and an unknown phospholipid. Strains KMM 330^T and KMM 331 contain C_{16 : 0} (20 and 17·6 %, respectively), C_{16 : 1}ω9c (23·5 and 23·0 %) and C_{18 : 1} (41·7 and 44·9 %) as major fatty acids, as well as C_{12 : 0} (6·9 and 4·9 %). C_{12 : 1}, iso-C_{13 : 0}, C_{13 : 0}, C_{13 : 1}, C_{14 : 0}, C_{14 : 1}ω7c, C_{14 : 0} 3-OH, C_{15 : 0}, iso-C_{15 : 0}, iso-C_{16 : 0}, iso-C_{17 : 0}, C_{12 : 0} 3-OH, C_{17 : 1}ω8c, C_{17 : 0}, cyc-C_{17 : 0} and C_{18 : 0} are present at 1 %. The G+C content of the DNA is 61·161·5 mol% (determined by the thermal denaturation method).

The type strain, KMM 330^T (=JCM 12285^T=NRIC 0583^T=CCUG 46540^T), was isolated from the sponge Pachastrella sp., collected from the Philippine Sea at a water depth of 750 m.