International Journal of Systematic and Evolutionary Microbiology (2014), 64, 3565–3570
DOI 10.1099/ijs.0.062935-0
Pseudomonas salegens sp. nov., a halophilic member of the genus Pseudomonas isolated from a wetland Mohammad Ali Amoozegar,1 Azadeh Shahinpei,2 Abbas Akhavan Sepahy,2,3 Ali Makhdoumi-Kakhki,4 Shima Sadat Seyedmahdi,2 Peter Schumann5 and Antonio Ventosa6 1
Correspondence Mohammad Ali Amoozegar
[email protected] or
[email protected]
Extremophiles Laboratory, Department of Microbiology, Faculty of Biology and Center of Excellence in Phylogeny of Living Organisms, College of Science, University of Tehran, Tehran, Iran
2
Microorganisms Bank, Iranian Biological Resource Centre (IBRC), ACECR Tehran, Iran
3
Department of Microbiology, Faculty of Biological Sciences, Islamic Azad University North Tehran Branch, Tehran, Iran
4
Department of Biology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran
5
Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstraße 7B, 38124 Braunschweig, Germany
6
Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain
A novel Gram-stain-negative, aerobic, non-endospore-forming, non-pigmented, rod-shaped, slightly halophilic bacterium, designated GBPy5T, was isolated from aquatic plants of the Gomishan wetland, Iran. Cells of strain GBPy5T were motile. Growth occurred with between 1 and 10 % (w/v) NaCl and the isolate grew optimally with 3 % (w/v) NaCl. The optimum pH and temperature for growth of the strain were pH 8.0 and 30 6C, respectively, while it was able to grow over a pH range of 6.5–9.0 and a temperature range of 4–35 6C. Phylogenetic analysis, based on 16S rRNA gene sequences, revealed that strain GBPy5T is a member of the genus Pseudomonas forming a monophyletic branch. The novel strain exhibited 16S rRNA gene sequence similarity of 95.4 % with type strains of Pseudomonas guariconensis PCAVU11T and Pseudomonas sabulinigri J64T, respectively. The major cellular fatty acids of the isolate were C18 : 1v7c (37.8 %), C16 : 0 (14.9 %), C16 : 1v7c (12.9 %), C12 : 0 3-OH (7.1 %) and C12 : 0 (7.0 %). The polar lipid pattern of strain GBPy5T comprised phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and one phospholipid. Ubiquinone 9 (Q-9) was the predominant lipoquinone. The G+C content of the genomic DNA of strain GBPy5T was 59.2 mol%. On the basis of the phenotypic and phylogenetic data, strain GBPY5T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas salegens sp. nov. is proposed. The type strain is GBPy5T (5IBRC-M 10762T5CECT 8338T).
The genus Pseudomonas, which was first described by Migula (1894), accommodates a group of Gram-stainnegative bacteria that are non-endospore-forming, aerobic, motile and rod-shaped (Palleroni, 2005). Pseudomonads are well-known micro-organisms with versatile metabolic activity and have been isolated from various environments The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain GBPy5T is KF830707. Three supplementary figures are available with the online version of this paper.
062935 G 2014 IUMS
Printed in Great Britain
like soil, plants, freshwater, clinical specimens and marine habitats (Palleroni, 1992; Romanenko et al., 2005). The taxonomic position of members of the genus Pseudomonas has changed several times (Stanier et al., 1966; Palleroni & Doudoroff, 1972; De Ley, 1992; Anzai et al., 2000). At the time of writing (October 2013) the genus comprises 207 species with validly published names (www.bacterio.net/m/ marinobacter.html; Euze´by, 1997). Several species of the genus Pseudomonas can tolerate NaCl concentrations of up to 8 % (w/v) and are considered to be halotolerant. For a few species, which were first described as members of the 3565
M. A. Amoozegar and others
genus Pseudomonas, it was reported that sodium ions are necessary for growth; Pseudomonas elongata (Humm, 1946) was later transferred to the genus Microbulbifer as Microbulbifer elongatus (Yoon et al., 2003) and Pseudomonas halophila (Fendrich 1988) has not yet had its taxonomic position defined (Anzai et al., 2000). These two are the only halophilic members of the genus Pseudomonas so far reported, based on the definition of Kushner & Kamekura (1988). Here we describe the isolation and polyphasic characterization of a novel, slightly halophilic microorganism isolated from a saline wetland in Iran and propose that this strain represents a novel species of the genus Pseudomonas. Strain GBPy5T was isolated from aquatic plants from the Gomishan saline wetland (pH 8.5–9.3, salinity 3–5 %) in Iran (37u 039 N 54u 019 E). The wetland is an alkaline thalassohaline, coastal–marine wetland, located along the eastern shore of the Caspian Sea in Iran (37u 049 06.39 N 54u 009 24.59 E). It has an area of about seventeen thousand hectares and is 27 m below sea level. Modified alkaliphilic halophile agar (MAHA) was used for the isolation procedure. It contained (g l21): NaCl 30, KCl 0.08, MgSO4 . 7H2O 0.04, FeSO4 . 7H2O 0.002, MnCl2 . 4H2O 0.0036, peptone 5, yeast extract 2, meat extract 1 and trisodium citrate 0.12; 1.5 % (w/v) agar was added when necessary. The pH of the medium was adjusted to pH 9.5 after supplementing the medium with (g l21): Na2CO3 10.6 and NaHCO3 8.42, which were autoclaved separately. Inoculated plates were incubated aerobically at 34 uC for up to 1 week. After successive cultivations, a pure isolate, designated strain GBPy5T, was obtained and routinely grown on 3 % (w/v) salt MH medium agar (Ventosa et al., 1982), pH 8 and 30 uC. Characterization of this strain was achieved by using a polyphasic approach, including analysis of conventional phenotypic features, chemotaxonomic data (polar lipid, fatty acid and quinone composition) and 16S rRNA gene sequences. The type species of the genus Pseudomonas, Pseudomonas aeruginosa IBRC-M 10828T, was obtained from the Iranian Biological Resource Center and used as the reference strain for comparison. It was cultured according to the recommendations of the culture collection. Genomic DNA of the isolate was extracted with a DNA extraction kit (High Pure PCR Template Preparation kit; Roche) according to the manufacturer’s protocol and the 16S rRNA gene was amplified using the bacterial universal primers, 27F and 1492R (Lane et al., 1985). Direct sequence determination of the PCR-amplified DNA was conducted on an ABI 3730XL DNA sequencer at Macrogen (Seoul, South Korea). 16S rRNA gene sequence analysis was performed with the ARB software package (Ludwig et al., 2004). The 16S rRNA gene sequence was aligned with published sequences of closely related bacteria and the alignment was confirmed and checked against both primary and secondary structures of the 16S rRNA molecule using the alignment tool of the ARB software package. Phylogenetic 3566
analysis was performed using the software package MEGA version 5 (Tamura et al., 2011) after obtaining multiple alignments of data available from public databases using CLUSTAL X (Thompson et al., 1997). Clustering was performed using the neighbour-joining (Saitou & Nei, 1987), minimum-evolution (Rzhetsky & Nei, 1992) and maximumlikelihood (Felsenstein, 1981) methods. Bootstrap analysis was used to evaluate the tree topology of the neighbourjoining data by performing 1000 resamplings (Felsenstein, 1985). Gene sequence similarity among species of the genus Pseudomonas was calculated using the Pairwise-Distance computing function of MEGA5. An almost complete 16S rRNA gene sequence of strain GBPy5T (1419 nt) was obtained. 16S rRNA gene sequence analysis revealed that strain GBPy5T is a member of the genus Pseudomonas. However, the novel strain exhibited levels of 16S rRNA gene sequence similarity equal to, or lower than, 95.4 % with type strains of all species of the genus Pseudomonas. The closest relatives of strain GBPy5T were Pseudomonas guariconensis PCAVU11T, Pseudomonas sabulinigri J64T and Pseudomonas bauzanensis BZ93T with a sequence similarity of 95.4 %, 95.4 % and 95.3 %, respectively. Phylogenetic analysis using the neighbour-joining algorithm revealed that the novel strain clustered with members of the genus Pseudomonas, although in a separate clade (Fig. 1). The phylogenetic position was confirmed in trees generated using the minimum-evolution and maximum-likelihood algorithms (Figs S1 and S2; available in the online Supplementary Material). Cell morphology of cells, from exponentially growing cultures, was examined using an Olympus BX41 microscope equipped with phase-contrast optics. Gram staining was performed using the Burke method (Murray et al., 1994) and the result was contrasted with the KOH test (Baron & Finegold, 1990). Physiological tests were conducted using 3 % (w/v) salt MH broth or agar medium, unless stated otherwise. Broth cultures were incubated at 30 uC in an orbital incubator at 150 r.p.m. Growth was monitored from the turbidity at OD600 using a spectroscopic method (model UV-160 A; Shimadzu). To determine the optimal temperature and pH for the growth of the strain, broth cultures were incubated at 4 uC and at 5– 40 uC (at intervals of 5.0 uC). The pH of the medium was varied between pH 5–10 at intervals of 0.5 pH unit. pH values of below 6, pH 6–9 and above 9 were obtained using sodium acetate/acetic acid, Tris/HCl and glycine/sodium hydroxide buffers instead of Na2CO3 and NaHCO3 in 3 % salt MH medium, respectively. Growth at different NaCl contents (0.05, 1, 2.5, 5, 7.5, 10, 12.5 and 15 %, w/v) was tested on MH medium at pH 8.0. Growth under anaerobic conditions was determined in MH broth medium at 30 uC for 20 days using an anaerobic chamber. The production of pyocyanin and of fluorescent pigments was tested using King A medium and King B medium, respectively (King et al., 1954). The presence of endospores was investigated using the Schaeffer–Fulton staining method (Murray et al., 1994). International Journal of Systematic and Evolutionary Microbiology 64
Pseudomonas salegens sp. nov.
Pseudomonas segetis FR1439T (AY770691) Pseudomonas marincola KMM 3042T (AB301071) Pseudomonas cuatrocienegasensis 1NT (EU791281) Pseudomonas oleovorans subsp. lubricantis RS1T (DQ842018) Pseudomonas taiwanensis BCRC 17751T (EU103629) Pseudomonas guariconensis PCAVU11T (HF674459) 98 84 Pseudomonas pohangensis H3-R18T (DQ339144) Pseudomonas aeruginosa LMG 1242T (Z76651) 94 Pseudomonas panipatensis Esp-1T (EF424401) Pseudomonas pachastrellae KMM 330T (AB125366) Pseudomonas salegens GBPy5T (KF830707) Pseudomonas xinjiangensis S3-3T (EU286805) Pseudomonas sabulinigri J64T (EU143352) Pseudomonas pelagia CL-AP6T (EU888911) 74 Pseudomonas litoralis 2SM5T (FN908483) 75 Pseudomonas bauzanensis BZ93T (GQ161991) 99 Marinimicrobium koreense M9T (AY839869.2) 80
0.01
98
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationship of strain GBPy5T with other members of the genus Pseudomonas. Accession numbers of sequences are given in parentheses. The sequence of Marinimicrobium koreense M9T (AY839869) was used as an outgroup. Bootstrap values (%) are based on 1000 replicates. Bar, 0.01 substitutions per nucleotide position.
Motility was analysed by the wet-mount method (Murray et al., 1994). Tests for catalase, oxidase, nitrate reduction, hydrolysis of aesculin and production of indole were carried out as recommended by Smibert & Krieg (1994). The hydrolysis of Tweens 20 and 80 was examined as described by Harrigan & McCance (1976). Antibiotic susceptibility tests were performed on Mueller–Hinton agar plus 3 % (w/v) sea salts (Ventosa et al., 1982) seeded with a bacterial suspension containing 1.56106 c.f.u. ml21 using discs (HiMedia) impregnated with various antimicrobial compounds. The plates were incubated at 30 uC for 48 h and the inhibition zone was interpreted according to the manufacturer’s manual. Other physiological and biochemical tests were performed as described previously (Ventosa et al., 1982; Quesada et al., 1984; Mata et al., 2002).
(30 mg) and tetracycline (30 mg). It was resistant to bacitracin (10 U), cefalotin (30 mg) and novobiocin (30 mg). Other phenotypic features are presented in Table 1 and in the species description.
Strain GBPy5T was Gram-stain-negative, strictly aerobic, catalase- and oxidase-positive, but did not produce endospores. Cells were rods and 0.3–0.6 mm60.8–1.9 mm. When grown for 3 days at 30 uC on 3 % MH medium, colonies were tiny, punctiform, convex, entire, smooth and non-pigmented with a diameter of 0.5 mm. This isolate was slightly halophilic, growing in media containing 1 to 10 % (w/v) NaCl and optimally in media containing 3 % (w/v) NaCl. No growth was observed in the absence of NaCl. The pH range for growth was 6.5–9.0 (optimum growth at pH 8.0) and the isolate grew at 4–35 uC (optimum growth at 30 uC). The type strain was susceptible to the following antimicrobial compounds: ampicillin (10 mg), carbenicillin (100 mg), cefoxitin (30 mg), chloramphenicol (30 mg), erythromycin (15 mg), gentamicin (10 mg), kanamycin (30 mg), neomycin (10 mg), nitrofurantoin (300 mg), polymyxin B (300 U), rifampicin (5 mg), streptomycin (10 mg), tobramycin (10 mcg), penicillin G (10 U), amikacin
Cell biomass for fatty acid, isoprenoid quinone and polar lipid analyses was obtained by cultivation in 3 % salt MH broth medium at 150 r.p.m. and 30 uC. Cells were harvested in the mid-exponential growth phase. The whole-cell fatty acid composition of strain GBPy5T was determined according to the standard protocol of the Microbial Identification System (Sherlock version 6.1; MIDI). Extracts were analysed using a Hewlett Packard model HP6890A gas chromatograph equipped with a flame-ionization detector, as described by Ka¨mpfer & Kroppenstedt (1996). Fatty acid peaks were identified using the TSBA40 database. The fatty acid profile of strain GBPy5T was characterized by the presence of the following fatty acids: C18 : 1v7c (37.8 %), C16 : 0 (14.9 %), summed feature 3 (C16 : 1v7c) (12.9 %), C12 : 0 3-OH (7.2 %), C12 : 0 (7.0 %), C19 : 0 cyclov8c (5.8 %), C10 : 0 3-OH (5.2 %), C18 : 0 (1.1 %) and C10 : 0 (1.0 %). The fatty acid composition of strain GBPy5T could not be compared to that of P.
http://ijs.sgmjournals.org
For determination of DNA base composition, DNA was isolated using a French pressure cell (Thermo Spectronic) and was purified by chromatography on hydroxyapatite as described by Cashion et al. (1977). The DNA G+C content was determined by reversed-phase HPLC of nucleosides according to Mesbah et al. (1989). The G+C content of the DNA of strain GBPy5T was 59.2 mol%. This value is within the range for other species of the genus Pseudomonas (58–69 mol%) (Palleroni, 2005), but much lower than that of P. guariconensis, the closest phylogenetically related species (Table 1).
3567
M. A. Amoozegar and others
Table 1. Differential characteristics of strain GBPy5T and phylogenetically related species of the genus Pseudomonas Strains: 1, GBPy5T (Pseudomonas salegens sp. nov.); 2, P. aeruginosa IBRC-M 10828T; 3, P. guariconensis IBRC-M 10856T; 4, P. sabulinigri IBRC-M 10839T; 5, Pseudomonas pachastrellae KMM 330T (Romanenko et al., 2005); 6, Pseudomonas xinjiangensis S3-3T (Liu et al., 2009). +, Positive; 2, negative; W, weak; NA, data not available. Data from this study, unless otherwise indicated. Characteristic Habitat Pigmentation Cell size (mm) Growth at (uC): 4 41 NaCl requirement Tolerance to 8 % (w/v) NaCl Reduction of nitrate to nitrite Utilization of: D-Glucose Maltose Xylose Fructose Glycerol D-Mannitol Acetate D-Alanine L-Asparagine DNA G+C content (mol%)
1
2
Wetland Clinical specimens ” + 0.3–0.660.8–1.9 0.2–0.661.2
3
4
5
6
Rhizospheric soil + 0.8–0.961.9–3.1
Black sand + 0.7–1.061.5–2.0
Deep-sea sponge ” 0.4–0.561.4–1.6
Desert sand + 0.6–0.760.9–1.8
+ ” ” +
” + ” +
+ + ” ”
”
+
” ” ” ” ” ” 2 + + 61.1
” ” ” ” ” ”
+ ” + +
” + ” ”
” + ” ”
+
+
”
” ” ” ” ” ” + 2 2 59.2
+ ” ” + + + + + + 67.0a*
+ ” + W
+ ” 2 + + 61.5b
” ” ” ” ” ” 2 2 + 58.1c
NA NA NA
60.9
*Data taken from: a, Palleroni (2005); b, Toro et al. (2013); c, Kim et al. (2009).
guariconensis PCAVU11T under standardized conditions because of the differing conditions they require for optimal growth. They each grow over a different range of NaCl concentrations and require different NaCl concentrations for optimum growth, so it was not possible to compare them using optimal growth conditions for both strains. However, like other species of the genus Pseudomonas, the hydroxylated fatty acids C10 : 0 3-OH, C12 : 0 3-OH and C12 : 0 2-OH were found in strain GBPy5T (Palleroni, 2005). The two predominant fatty acids of GBPy5T were C18 : 1v7c and C16 : 0, which have also been found in P.aeruginosa, the type species of the genus, and in the most closely related species, P. guariconensis (Toro et al., 2013).
detected in strain GBPy5T were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and one phospholipid (Fig. S3), whereas P. aeruginosa IBRC-M 10828T contained phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, two unknown aminophospholipids and two phospholipids. Strain GBPy5T differs from P. guariconensis IBRC-M 10856T and P. sabulinigri IBRC-M 10839T as the closest relative strains, by the absence of unknown aminophospholipids. The major quinone present was ubiquinone 9 (Q-9). The respiratory lipoquinone of strain GBPy5T was typical of that found in members of the genus Pseudomonas (Romanenko et al., 2005; Liu et al., 2009; Toro et al., 2013).
The polar lipids and respiratory quinones of strain GBPy5T were analysed as described by Groth et al. (1996). Polar lipid analysis of strain GBPy5T was performed by twodimensional TLC. Total lipids were visualized by spraying with molybdophosphoric acid and heating at 150 uC for 10 min and further characterized by spraying with specific reagents, including ninhydrin spray reagent, and heating at 100 uC for 10 minutes (aminolipids). Other spray reagents used were molybdenum blue (phospholipids) and anaphthol/sulfuric acid (glycolipids). The polar lipids
In conclusion, the results obtained from the polyphasic studies indicate that strain GBPy5T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas salegens sp. nov. is proposed.
Pseudomonas salegens (sal.e9gens. L. n. sal salis salt; L. part. adj. egens being in need; N.L. part. adj. salegens being in need of salt).
3568
International Journal of Systematic and Evolutionary Microbiology 64
Description of Pseudomonas salegens sp. nov.
Pseudomonas salegens sp. nov.
Cells are Gram-stain-negative, and motile rods, 0.8– 1.960.3–0.6 mm. Endospores are not produced. Colonies are tiny, punctiform, convex, entire, smooth and nonpigmented with a diameter of 0.5 mm on 3 % MH agar medium after 72 h of incubation at 30 uC. Cells are nonfluorescent on King A and B media. Strictly aerobic. Slightly halophilic, growing between NaCl concentrations of 1 and 10 % (w/v), with optimal growth at 3 % (w/v) NaCl. No growth occurs in the absence of NaCl. Grows at 4–35 uC (optimally at 30 uC) and pH 6.5–9.0 (optimally at pH 8.0). Catalase- and oxidase-positive. Tweens 20 and 80 and DNA are hydrolysed, while casein, aesculin and starch are not. Nitrate is reduced, nitrite is not. Indole or H2S are not produced. Acid is not produced from various carbohydrate sources. The following compounds are utilized as sole sources of carbon and energy: acetate, fumarate, sebacic acid, hydroxybutyrate and propionate. The following compounds are not utilized as a sole source of carbon and energy: L-arabinose, cellobiose, D-galactose, D-glucose, lactose, maltose, D-mannitol, D-mannose, Draffinose, L-rhamnose, D-ribose, trehalose, D-xylose, Lalanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-leucine, L-methionine, L-ornithine, L-serine and L-tyrosine. Polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and one phospholipid. The major isoprenoid quinone is ubiquinone 9 (Q-9). The predominant fatty acids are C18 : 1v7c, C16 : 0 and C16 : 1v7c. The type strain is GBPy5T (5IBRC-M 10762T5CECT 8338T); it was isolated from an aquatic plant in the Gomishan wetland in Iran. The DNA G+C content of the type strain is 59.2 mol%.
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a
maximum likelihood approach. J Mol Evol 17, 368–376. Felsenstein, J. (1985). Confidence limits on phylogenies: an approach
using bootstrap. Evololution 39, 783–791. Fendrich, C. (1988). Halovibrio variabilis gen. nov. sp. nov., Pseudomonas halophila sp. nov. and a new halophilic aerobic coccoid eubacterium from Great Salt Lake, Utah, USA. Syst Appl Microbiol 11, 36–43. Groth, I., Schumann, P., Weiss, N., Martin, K. & Rainey, F. A. (1996).
Agrococcus jenensis gen. nov., sp. nov., a new genus of actinomycetes with diaminobutyric acid in the cell wall. Int J Syst Bacteriol 46, 234– 239. Harrigan, W. F. & McCance, M. E. (1976). Laboratory Methods in Food
and Dairy Microbiology. London: Academic Press. Humm, H. J. (1946). Marine agar-digesting bacteria of the South
Atlantic coast. Bull Duke Univ Mar Sta 3, 45–75. Ka¨mpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of
fatty acid patterns of coryneform bacteria and related taxa. Can J Microbiol 42, 989–1005. Kim, K. H., Roh, S. W., Chang, H. W., Nam, Y. D., Yoon, J. H., Jeon, C. O., Oh, H. M. & Bae, J. W. (2009). Pseudomonas sabulinigri sp. nov.,
isolated from black beach sand. Int J Syst Evol Microbiol 59, 38–41. King, E. O., Ward, M. K. & Raney, D. E. (1954). Two simple media for
the demonstration of pyocyanin and fluorescin. J Lab Clin Med 44, 301–307. Kushner, D. J. & Kamekura, M. (1988). Physiology of halophilic
eubacteria. In Halophilic Bacteria, vol. I, pp. 109–140. Edited by F. Rodriguez-Valera. Boca Raton, FL: CRC Press. Lane, D. J., Pace, B., Olsen, G. J., Stahl, D. A., Sogin, M. L. & Pace, N. R. (1985). Rapid determination of 16S ribosomal RNA sequences
for phylogenetic analyses. Proc Natl Acad Sci U S A 82, 6955–6959. Liu, M., Luo, X., Zhang, L., Dai, J., Wang, Y., Tang, Y., Li, J., Sun, T. & Fang, C. (2009). Pseudomonas xinjiangensis sp. nov., a moderately
thermotolerant bacterium isolated from desert sand. Int J Syst Evol Microbiol 59, 1286–1289.
Acknowledgements We thank J. Euze´by for his help with the etymology of the new taxon. This work was supported by grants from Iranian Biological Resource Centre (IBRC) (MI-1391-15) (to M. A. A.), from the Spanish Ministerio de Economı´a y Competitividad (CGL2010-19303 and CGL2013-46941-P) that include European Funds (FEDER) and Junta de Andalucı´a (P10-CVI-6226) (to A. V.).
Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar, Buchner, A., Lai, T., Steppi, S. & other authors (2004). ARB: a software environment for sequence data. Nucleic Acids
Res 32, 1363–1371. Mata, J. A., Martı´nez-Ca´novas, J., Quesada, E. & Be´jar, V. (2002). A
detailed phenotypic characterisation of the type strains of Halomonas species. Syst Appl Microbiol 25, 360–375. Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise
References Anzai, Y., Kim, H., Park, J. Y., Wakabayashi, H. & Oyaizu, H. (2000).
Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence. Int J Syst Evol Microbiol 50, 1563–1589. Baron, E. J. & Finegold, S. M. (1990). Bailey and Scott’s Diagnostic
Microbiology, 8th edn. St Louis, MO: Mosby. Cashion, P., Holder-Franklin, M. A., McCully, J. & Franklin, M. (1977).
A rapid method for the base ratio determination of bacterial DNA. Anal Biochem 81, 461–466. De Ley, J. (1992). The proteobacteria: ribosomal RNA cistron
similarities and bacterial taxonomy. In The Prokaryotes, 2nd edn, pp. 2111–2140. Edited by A. Balows, H. G. Tru¨per, M. Dworkin, W. Harder & K. H. Schleifer. New York: Springer. Euze´by, J. P. (1997). List of bacterial names with standing in
nomenclature: a folder available on the Internet. Int J Syst Bacteriol 47, 590–592. http://www.bacterio.net http://ijs.sgmjournals.org
measurement of the G+C content of deoxyribonucleic acid by highperformance liquid chromatography. Int J Syst Evol Bacteriol 39, 159– 167. ¨ ber ein neues system der bakterien. Arb Bakteriol Migula, W. (1894). U Inst Technisch Hochsch Karlsruhe 1, 235–238 (in German). Murray, R. G. E., Doetsch, R. N. & Robinow, C. F. (1994).
Determinative and cytological light microscopy. In Methods for General and Molecular Bacteriology, pp. 21–41. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology. Palleroni, N. J. (1992). Introduction to the family Pseudomonadaceae.
In The Prokaryotes, 2nd edn, pp. 3071–3085. Edited by A. Balows, H. G. Truper, U. Umlaut, M. Dworkin, W. Harder & K. H. Schleifer. New York: Springer. Palleroni, N. J. (2005). Genus I. Pseudomonas Migula 1894, 237AL. In
Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2, part B, pp. 323–379. Edited by D. J. Brenner, N. R. Krieg, J. T. Staley & G. M. Garrity. New York: Springer. 3569
M. A. Amoozegar and others Palleroni, N. J. & Doudoroff, M. (1972). Some properties and subdivisions
of the genus Pseudomonas. Annu Rev Phytopathol 10, 73–100.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using
Quesada, E., Ventosa, A., Ruiz-Berraquero, F. & RamosCormenzana, A. (1984). Deleya halophila, a new species of moderately
maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739.
halophilic bacteria. Int J Syst Bacteriol 34, 287–292.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible
Romanenko, L. A., Uchino, M., Falsen, E., Frolova, G. M., Zhukova, N. V. & Mikhailov, V. V. (2005). Pseudomonas pachastrellae sp. nov.,
isolated from a marine sponge. Int J Syst Evol Microbiol 55, 919–924. Rzhetsky, A. & Nei, M. (1992). A simple method for estimating and
testing minimum-evolution trees. Mol Biol Evol 9, 945–967. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425. Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In
Methods for General and Molecular Bacteriology, pp. 607–654. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology.
strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882. Toro, M., Ramı´rez-Bahena, M. H., Cuesta, M. J., Vela´zquez, E. & Peix, A. (2013). Pseudomonas guariconensis sp. nov., isolated from rhizo-
spheric soil. Int J Syst Evol Microbiol 63, 4413–4420. Ventosa, A., Quesada, E., Rodrı´guez-Valera, F., Ruiz-Berraquero, F. & Ramos-Cormenzana, A. (1982). Numerical taxonomy of moder-
ately halophilic Gram-negative rods. J Gen Microbiol 128, 1959– 1968. Yoon, J. H., Kim, H., Kang, K. H., Oh, T. K. & Park, Y. H. (2003).
pseudomonads: a taxonomic study. J Gen Microbiol 43, 159–271.
Transfer of Pseudomonas elongata Humm 1946 to the genus Microbulbifer as Microbulbifer elongatus comb. nov. Int J Syst Evol Microbiol 53, 1357–1361.
3570
International Journal of Systematic and Evolutionary Microbiology 64
Stanier, R. Y., Palleroni, N. J. & Doudoroff, M. (1966). The aerobic
