International Journal of Systematic and Evolutionary Microbiology (2013), 63, 3168–3174
DOI 10.1099/ijs.0.049452-0
Pseudomonas formosensis sp. nov., a gammaproteobacteria isolated from food-waste compost in Taiwan Shih-Yao Lin,1 Asif Hameed,1 You-Cheng Liu,1 Yi-Han Hsu,1 Wei-An Lai1,2 and Chiu-Chung Young1,2 Correspondence Chiu-Chung Young
[email protected]
1
Department of Soil and Environmental Sciences, College of Agriculture and Natural Resources, National Chung Hsing University, Taichung, Taiwan, ROC
2
Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan
A taxonomic study was carried out on a novel aerobic bacterial strain, designated CC-CY503T, isolated from food-waste compost in Taiwan. Cells were Gram-stain-negative short rods, motile by means of a monopolar flagellum. Strain CC-CY503T was able to grow at 20–50 6C and pH 6.0–10.0 and to tolerate ,6 % NaCl (w/v). Phylogenetic analysis of 16S rRNA gene sequences showed that this bacterium belonged to the genus Pseudomonas, with Pseudomonas pertucinogena ATCC 190T as the closest neighbour, sharing a sequence similarity of 97.9 %. The DNA–DNA relatedness value of strain CC-CY503T with P. pertucinogena ATCC 190T was 37.8±2.3 %. The phylogenetic trees reconstructed based on gyrB and rpoB gene sequences supported the classification of strain CC-CY503T as a novel member of the genus Pseudomonas. The predominant quinone system was ubiquinone (Q-9) and the DNA G+C content was 63.1±0.4 mol%. The major fatty acids were C12 : 0, C16 : 0, C17 : 0 cyclo, C19 : 0 cyclo v8c and summed features 3 and 8 consisting of C16 : 1v7c/C16 : 1v6c and C18 : 1v7c/C18 : 1v6c, respectively. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and phosphatidylcholine. On the basis of its distinct phylogenetic, phenotypic and chemotaxonomic features, strain CC-CY503T (5BCRC 80437T5JCM 18415T) is proposed to represent a novel species within the genus Pseudomonas, for which the name Pseudomonas formosensis sp. nov. is proposed.
The genus Pseudomonas was first described by Migula (1894) and the members of this genus are widespread micro-organisms that have been isolated from a variety of natural sources, including soil, plants and mineral waters, and from clinical specimens, and they are characterized by a high level of metabolic diversity (Rossello´ et al., 1991; Moore et al., 1996). Additionally, groups of species differ considerably in their nutritional adaptability and serve a variety of functions such as decomposition of organic matter and plant growth-promotion and can also act as pathogens (Palleroni, 1993; Peix et al., 2009). The classification of the genus Pseudomonas has undergone multiple reassessments on the basis of physiological, molecular and phenotypic features (Sneath et al., 1981), DNA–DNA hybridization (Palleroni, 1984), 16S rRNA The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, gyrase subunit B (gyrB) and polymerase beta subunit (rpoB) gene sequences of Pseudomonas formosensis CC-CY503T are JF432053, JQ864235 and JQ864236, respectively. Four supplementary figures are available with the online version of this paper.
3168
gene sequence similarity (Anzai et al., 2000) and chemotaxonomic data (Oyaizu & Komagata, 1983; Vancanneyt et al., 1996). In general, species of the genus Pseudomonas are recognized as a repository for strictly aerobic Gramnegative rods that are motile by means of single or several polar flagella. While investigating the bacterial flora inhabiting foodwaste compost, strain CC-CY503T was isolated in Taiwan. Briefly, a sample of food-waste compost (10 g) was introduced into sterile water and shaken at 30 uC for two days. Subsequently, this sample was serially diluted (10fold dilutions each), spread plated (100 ml plate21) on nutrient agar (NA; Hi-Media) and incubated in the dark for two days. A pale yellow colony appeared and was picked up, purified and subcultured on NA. The purified bacterial strain, designated CC-CY503T, was preserved as glycerol suspension at 280 uC for further characterization. For taxonomic purposes, reference strains were purchased from their respective culture collection centres. For direct comparative analysis, all these strains were grown on NA at 30 uC for two days, unless specified otherwise. 049452 G 2013 IUMS Printed in Great Britain
Pseudomonas formosensis sp. nov.
Bacterial genomic DNA was isolated by using an UltraClean Microbial Genomic DNA Isolation kit (MO BIO) by following the manufacturer’s instructions. The extracted DNA was used as template to amplify the 16S rRNA gene. The PCR was performed with bacterial universal primers 1F (59-GAGTTTGATCATGGCTCAGA-39) and 9R (59-AAGGAGGTGATCCAACCGCA-39). Primers 3F (59-CCTACGGGAGGCAGCAG-39), 5F (59-AAACTCAAATGAATTGACGGGG-39) and 4R (59-TTACCGCGGCTGCTGGCAC-39) were used for sequencing reactions (Hung et al., 2005). The gyrB gene was amplified by PCR using primers UP-1 (59-CAYGCNGGNGGNAARTTYGA-39) and UP-2r (59-CCRTCNACRTCNGCRTCNGTCAT-39) as described by Yamamoto & Harayama (1995). The rpoB gene, a highly conserved housekeeping gene that encodes the RNA polymerase b subunit, has been used for phylogenetic analysis. The primers LAPS (59-TGGCCGAGAACCAGTTCCGCGT-39) and LAPS27 (59-CGGCTTCGTCCAGCTTGTTCAG-39) were used to amplify about 1.2 kb of rpoB (Ait Tayeb et al. 2005). Gene sequencing was performed by using the Bigdye terminator kit (Heiner et al., 1998), and nucleotide sequences of PCR products were determined using an automatic DNA sequencer (ABI PRISM 310; Applied Biosystems) (Watts & MacBeath, 2001). The DNA sequences were then assembled using the Fragment Assembly System program from the Wisconsin package (GCG, 1995). The almost-complete 16S rRNA gene sequence (1493 bp) of strain CC-CY503T was uploaded to the EzBioCloud (EzTaxon-e Database; Kim et al., 2012) and NCBI servers for BLAST search. Subsequently, closely related 16S rRNA gene sequences were retrieved from EzTaxon-e, Ribosomal Database Project (Maidak et al., 2001) or GenBank and aligned by using the CLUSTAL X (1.83) program (Thompson et al., 1997). For further determination of phylogenetic structure of the genus Pseudomonas, partial gyrB and rpoB gene sequences were analysed. The phylogenetic analyses were performed using MEGA 5 software (Tamura et al., 2011) and the topologies of the resultant neighbour-joining trees were evaluated by bootstrap analyses (Felsenstein, 1985) after 1000 replications. Comparative 16S rRNA, gyrB and rpoB gene sequence analyses positioned the novel strain in the genus Pseudomonas as an independent lineage. In 16S rRNA gene sequence analyses, strain CC-CY503T shared a high pairwise sequence similarity to Pseudomonas pertucinogena BCRC 80287T (5 ATCC 190T) (97.9 %), Pseudomonas litoralis CECT 7670T (97.0 %), Pseudomonas bauzanensis DSM 22558T (96.9 %), Pseudomonas xiamenensis JCM 13530T (96.7 %) and Pseudomonas pelagia JCM 15562T (95.5 %) and lower sequence similarity to others strains examined. Supporting evidence for the classification of strain CC-CY503T as a novel species has been provided by rpoB and gyrB gene sequence analysis. The pairwise sequence similarities (%) of each dataset were calculated using CLUSTAL X; the similarity of the rpoB gene http://ijs.sgmjournals.org
sequence of strain CC-CY503T to that of P. pertucinogena BCRC 80287T, P. xiamenensis JCM 13530T and P. pelagia JCM 15562T was 87.7, 88.2 and 83.0 %, respectively, whereas the similarity of the gyrB gene sequence of strain CC-CY503T to that of P. pertucinogena BCRC 80287T, P. xiamenensis JCM 13530T and P. pelagia JCM 15562T was 85.7, 87.8 and 79.3 %, respectively. Compared with 37 closed-related species of the genus Pseudomonas, the similarity values of rpoB and gyrB gene sequences of strain CC-CY503T ranged between 83.9 % and 100 % (rpoB) and 78.0 % and100 % (gyrB) at the interspecies level. Corresponding values obtained for strain CC-CY503T with regard to reference strains were almost within the reported threshold. The phylogenetic trees reconstructed by the neighbour-joining method using 16S rRNA, rpoB and gyrB gene sequences are shown in Fig. 1 and Figs. S1 and S2 (available in IJSEM Online), respectively. All these phylogenetic trees supported the affiliation of strain CCCY503T as a novel member of the genus Pseudomonas. DNA–DNA associations were conducted between strain CC-CY503T and P. pertucinogena BCRC 80287T. Chromosomal DNA of P. pertucinogena BCRC 80287T was used to construct hybridization probes by labelling with DIG–11-dUTP. Reciprocal DNA–DNA hybridization was performed by using DIG-labelled probe from strain CC-CY503T. For this purpose, bacterial genomic DNA samples were isolated by using UltraClean Microbial Genomic DNA Isolation kits (MO BIO) according to the manufacturer’s instructions. DNA samples from strain CCCY503T and P. pertucinogena BCRC 80287T were loaded onto positively charged membranes as described by Seldin & Dubnau (1985). The experiment was carried out in triplicate for each sample. The DNA–DNA relatedness value of P. pertucinogena BCRC 80287T with strain CCCY503T was 37.8±2.3 %. Reciprocal DNA–DNA hybridization values obtained for strain CC-CY503T with P. pertucinogena BCRC 80287T and P. bauzanensis DSM 22558T were 35.1±2.7 % and 48.1±1.7 %, respectively. Using the established molecular criteria for species-level relatedness (Wayne et al., 1987), strain CC-CY503T shows low DNA–DNA relatedness with other closely related related species, which supports its genomic distinction from members of the genus Pseudomonas. Colony morphology, presence of flagella and morphology of the cells of strain CC-CY503T were investigated using the colonies/cells grown on NA. Gram staining was performed as described by Murray et al. (1994). Cell morphology was studied by transmission electron microscopy (JEM-1400; JEOL) as well as by light microscopy (model A3000; Zeiss). Growth of CC-CY503T was also tested on tryptic soy agar (TSA; Difco) and R2A agar (Oxoid). Growth was tested using nutrient broth (NB; Hi-Media) at 20–50 uC (10 uC increments), pH 4.0–11.0 (1 unit increments) and NaCl concentrations of 1.0–5.0 % (1 % increments). Catalase activity was determined by assessing bubble production by 3169
S.-Y. Lin and others 100 Pseudomonas fuscovaginae MAFF 301177 (AB021381) Pseudomonas asplenii LMG 2137T (Z76655) 66 74 Pseudomonas vranovensis CCM 7279T (AY970951)
0.01
63
Pseudomonas japonica IAM 15071T (AB126621) Pseudomonas putida DSM 291T (Z76667) Pseudomonas oryzihabitans IAM 1568T (D84004) 72 52 Pseudomonas monteilii CIP 104883T (AF064458) 50
Pseudomonas plecoglossicida FPC951T (AB009457) 61 79 Pseudomonas mosselii CIP 105259T (AF072688) 57 Pseudomonas taiwanensis BCRC 17751T (EU103629) 91
100
Pseudomonas parafulva AJ 2129T (AB060132) Pseudomonas cremoricolorata NRIC 0181T (AB060136) Pseudomonas fulva IAM 1529T (D84015) Pseudomonas vancouverensis ATCC 700688T (AJ011507) Pseudomonas moraviensis CCM 7280T (AY970952)
99
Pseudomonas baetica a390T (FM201274)
Pseudomonas jessenii CIP 105274T (AF068259) 61 Pseudomonas reinekei MT-1T (AM293565) Pseudomonas argentinensis CH01T (AY691188) 100
Pseudomonas straminea IAM 1598T (D84023)
Pseudomonas stutzeri ATCC 17588T (CP002881) Pseudomonas alcaliphila AL15-21T (AB030583) 89 83 Pseudomonas oleovorans subsp. lubricantis RS1T (DQ842018) 56 95 Pseudomonas toyotomiensis HT-3T (AB453701) Pseudomonas mendocina LMG 1223T (Z76664)
98
Pseudomonas sagittaria CC-OPY-1T (JQ277453) Pseudomonas tuomuerensis 78-123T (DQ868767)
68 72 97
Pseudomonas alcaligenes LMG 1224T (Z76653) Pseudomonas aeruginosa LMG 1242T (Z76651)
76
99
Pseudomonas otitidis MCC10330T (AY953147) Pseudomonas benzenivorans DSM 8628T (FM208263) Pseudomonas pachastrellae KMM 330T (AB125366) 82
Pseudomonas pelagia CL-AP6T (EU888911) Pseudomonas sabulinigri J64T (EU143352)
55
Pseudomonas litoralis CECT 7670T (FN908483)
97 95
71
73 56
Pseudomonas pertucinogena IFO 14163T (AB021380) Pseudomonas formosensis CC-CY503T (JF432053) Pseudomonas xiamenensis C10-2T (DQ088664) Pseudomonas bauzanensis BZ93T (GQ161991) Cellvibrio ostraviensis LMG 19434T (AJ493583) Halomonas elongata ATCC 33173T (M93355)
Fig. 1. Phylogenetic analysis of strain CC-CY503T and other closely related strains of species of the genus Pseudomonas based on 16S rRNA gene sequences. Distances and clustering were determined by using the neighbour-joining method with the software package MEGA version 5. Bootstrap values (.50 %) based on 1000 replications are shown as percentages at the branching points. Bar, 0.01 substitutions per nucleotide position.
cells in 3 % (v/v) H2O2 and oxidase activity was determined by using 1 % (w/v) N,N,N,N,-tetramethyl-1,4-phenylenediamine reagent (bioMe´rieux). DNase testing was conducted by using DNase test agar (Hi-media). Carbon source utilization patterns were determined by using the Biolog GN2 MicroPlate (bioMe´rieux). Nitrate reduction,
indole production, activities of b-galactosidase and urease, and hydrolysis of aesculin and gelatin and of 12 substrates were tested with API 20 NE strips. The activities of various enzymes were determined by using the API ZYM system (bioMe´rieux) (Delisle et al., 1986). Antibiotic susceptibility was tested by using ATB PSE 5 strips (bioMe´rieux) by
3170
International Journal of Systematic and Evolutionary Microbiology 63
Pseudomonas formosensis sp. nov.
l21) and cotrimoxazole (2 mg l21). Additionally, strain CCCY503T showed several distinct physiological and biochemical characteristics which differentiated it from other species of the genus Pseudomonas, as given in Table 1.
following the manufacturer’s recommendations, the strips were incubated at 30 uC for 24 h. Strain CC-CY503T is a Gram-stain-negative, rod-shaped bacterium, which possesses a monopolar flagellum. Cells are 1.6–1.8 mm in length and 0.7–0.9 mm in diameter (Fig. S3). Colonies are circular, undulate, convex and pale yellow after 2 days of incubation on NA. In NB, strain CCCY503T can grow at 20–50 uC, at a pH range from 6.0 to 10.0, and can tolerate up to 5 % NaCl (w/v). In the biochemical tests, both strain CC-CY503T and P. pertucinogena ATCC 190T are positive for the following: catalase and oxidase activity; assimilation of capric acid, adipic acid and malic acid; utilization of Tween 40, Tween 80, pyruvic acid methyl ester, succinic acid monomethylester, acetic acid, b-hydroxybutyric acid, a-ketoglutaric acid, propionic acid, sebacic acid, succinic acid, bromosuccinic acid, L-alaninamide, D-alanine and L-alanine; activity of alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase; both are susceptible to ampicillin-sulbactam (4–8 mg l21), ticarcillin-pyo (64 mg l21), ticarcillin-clavulanic acid (16 mg l21), ticarcillin clavulanic acid-pyo (64 mg l21), imipenem (4–8 mg l21), meropenem (4–8 mg l21), amikacin (16–32 mg l21), gentamicin (4–8 mg l21), tobramycin (4–8 mg l21), ciprofloxacin (1–2 mg l21), colistin (2 mg
Polar lipids were extracted and analysed by two-dimensional TLC, and isoprenoid quinones were purified by the methods of Minnikin et al. (1984) and analysed by HPLC as described by Collins (1985). For analysis of DNA G+C content, a DNA sample was prepared and degraded enzymically into nucleosides as described by Mesbah et al. (1989). The nucleoside mixture obtained was then separated via HPLC. Fatty acid methyl esters were prepared, separated and identified according to the standard protocol (Paisley, 1996) of the Microbial Identification System (MIDI) (Sasser, 1990) by using a gas chromatograph (Agilent 7890A) fitted with a flameionization detector. The novel strain and the reference strains were grown on NA for 48 h at 30 uC. All the strains exhibited similar growth rates; exponential-phase harvests were subjected to saponification, methylation and extraction (Miller, 1982). Identification and comparison were made by using the Aerobe (RTSBA6) database of the MIDI System (Sherlock version 6.0). DNA G+C content of strain CC-CY503T was 63.1±0.4 mol%. The polar lipid profile of strain CC-CY503T
Table 1. Characteristics that differentiate strain CC-CY503T from the other closely related species of the genus Pseudomonas Strains: 1, CC-CY503T; 2, P. pertucinogena BCRC 80287T; 3, P. litoralis CECT 7670T; 4, P. bauzanensis DSM 22558T; 5, P. xiamenensis JCM 13530T; 6, P. aeruginosa ATCC 10145T. Data in column 3 was obtained from Pascual et al. (2012); Data in column 6 was obtained from Gibello et al. (2011), Colwell (1965) and Xiao et al. (2009). +, Positive; 2, negative; NA, no data available. Characteristic NaCl range (w/v) pH range Temperature range (uC) Nitrate reduction Assimilation (API 20NE) Malic acid Trisodium citrate Carbon sources (Biolog GN2) a-Ketobutyric acid b-Hydroxybutyric acid Citric acid DL-Lactic acid L-Glutamic acid D-Serine L-Threonine Enzymes (API ZYM) Alkaline phosphatase Lipase (C14) Cystine arylamidase Acid phosphatase DNA G+C content (mol%)
1
2
3
4
5
6
0–5.0 6.0–10.0 20–50 2
0–5.0 6.0–9.0 20–30 2
0–15 NA 15–37 2
0–10.0 6.0–10.0 6–37 +
0–8.0* NA 10–45* +
0–5.0 4.5–9.0 7–44 +
+ 2
+ 2
2 2
+ +
2 +
NA
2 + 2 2 2 2 2
+ + 2 2 + + +
2 2 2 + + 2 2
+ + + + + + 2
+ 2 2 + 2 2 2
NA
+ + + + 63.1±0.4
+ + 2 + 63.6±0.2
2 2 2 2
+ + 2 + 64.4±0.9
+ + 2 + 61.2*
2 + 2 + 67.2
NA
+
2 2 + + NA NA
*Data were obtained from Lai & Shao (2008). http://ijs.sgmjournals.org
3171
S.-Y. Lin and others
was similar to that of P. pertucinogena ATCC 190T with respect to the presence of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), which is in agreement with data published previously for species of the genus Pseudomonas (Pascual et al., 2012). In addition, a moderate amount of phosphatidylcholine (PC) was also detected irrespective of some quantitative difference in the two strains (Fig. S4). The predominant quinone system was ubiquinone (Q-9), which is compatible with findings for other species of the genus Pseudomonas. The major fatty acids in strain CC-CY503T were C12 : 0, C16 : 0, C17 : 0 cyclo, C19 : 0 cyclo v8c, C16 : 1v7c/C16 : 1v6c and C18 : 1v7c/C18 : 1 v6c which are generally present in members fo the genus Pseudomonas (Xiao et al., 2009; Pascual et al., 2012). Minor fatty acids are C10 : 0 3-OH, C12 : 0 3-OH and isoC15 : 0 3-OH. The details of the fatty acid profiles of strain CC-CY503T and other closely related species are given in Table 2. Based on the phylogenetic, morphological, biochemical and chemotaxonomic evidence, including 16S rRNA, gyrB and rpoB gene sequences, DNA–DNA association, carbon
Table 2. Comparison of the cellular fatty acid contents (%) of strain CC-CY503T and closely related species of the genus Pseudomonas Strains: 1, CC-CY503T; 2, P. pertucinogena BCRC 80287T; 3, P. litoralis CECT 7670T; 4, P. bauzanensis DSM 22558T; 5, P. xiamenensis JCM 13530T; 6, P. pelagia KCCM 90073T; 7, P. aeruginosa ATCC 10145T. Major fatty acids (.5 % of total fatty acids) are highlighted in bold type. TR, Trace (less than 1 %); NA, no data available. Data in columns 3 and 6 are from Pascual et al. (2012); data in column 7 was obtained from Xiao et al. (2009). Fatty acid Saturated C12 : 0 C14 : 0 C16 : 0 C17 : 0 Unsaturated C17 : 1v8c C19 : 0 cyclo v8c Branched iso-C17 : 0 C17 : 0 cyclo Hydroxy C10 : 0 3-OH C12 : 0 3-OH iso-C15 : 0 3-OH Summed features C16 : 1v7c/ C16 : 1v6c C18 : 1v7c/ C18 : 1v6c
3172
1
2
3
4
5
6
7.7
6.4
7.9
7.5
7.0
8.1
TR
TR
TR
TR
TR
13.1
10.3 2.1
18.5
TR
TR
15.2 14.2 TR
7
4.8 1.3 11.8 20.5 TR
TR
TR
TR
1.8 9.2
TR
TR
TR
1.8
15.5
6.4
1.1 1.3
NA
1.6 12.9
TR
TR
NA
8.9
13.8
1.8 5.7
TR
5.7
4.4
TR
3.8 3.7 3.0
4.5 4.0 2.4
3.0 4.2
3.3 3.8
3.1 4.4
3.6 4.5
NA
TR
3.2 3.6 1.3
NA
NA
10.6
9.7
18.1
5.1
5.9
24.6 20.0
36.9
23.7
32.6
29.7 34.5
35.4 38.9
TR
7.8 TR
TR
source utilization, substrate assimilation and enzyme activities, strain CC-CY503T is clearly distinct from P. pertucinogena BCRC 80287T, P. litoralis CECT 7670T and P. bauzanensis DSM 22558T with regard to several biochemical and molecular features. Therefore, we classify strain CC-CY503T as a representative of a novel species in the genus Pseudomonas, for which the name Pseudomonas formosensis sp. nov. is proposed. Description of Pseudomonas formosensis sp. nov. Pseudomonas formosensis [for.mo.sen9sis. N.L. fem. adj. formosensis of or pertaining to Formosa (Taiwan), the beautiful island]. Cells are Gram-stain-negative, rod-shaped, 1.6–1.8 mm in length and 0.7–0.9 mm in diameter and possess a monopolar flagellum. Grows well in solid and semi-solid media, but cannot grow under strictly anaerobic conditions. Colonies are circular, undulate, convex and pale yellow after 2 days of incubation on NA. In NB, the growth temperature ranges from 20 to 50 uC, pH 6.0–10.0 and the species tolerates ,6 % (w/v) NaCl. Oxidase- and catalasepositive. Cells can utilize Tween 40, Tween 80, pyruvic acid methyl ester, succinic acid monomethyl-ester, acetic acid, b-hydroxybutyric acid, a-ketoglutaric acid, propionic acid, sebacic acid, succinic acid, bromosuccinic acid, L-alaninamide, D-alanine and L-alanine as sole carbon sources. Nitrate and nitrite are not reduced; capric acid, adipic acid and malic acid are assimilated. Positive reactions for alkaline phosphatase, acid phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase and naphthol-ASBI-phosphohydrolase, but negative for trypsin, a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase. Sensitive to amikacin, ciprofloxacin, colistin, ampicillin-sulbactam, gentamicin, imipenem, meropenem, ticarcillin-clavulanic acid, ticarcillin clavulanic acid-pyo, ticarcillin-pyo, tobramycin and cotrimoxazole in ATB PSE 5 strips. The fatty acid profile consists of C12 : 0, C16 : 0, C17 : 0 cyclo, C19 : 0 cyclo v8c, C16 : 1v7c/C16 : 1 v6c and C18 : 1v7c/C18 : 1v6c. The polar lipid profile contains diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and phosphatidylcholine (PC). The predominant quinone is ubiquinone (Q-9). The type strain is CC-CY503T (5BCRC 80437T5JCM 18415T), isolated from food-waste compost, in Taiwan. The DNA G+C content of the type strain is 63.1±0.4 mol%.
Acknowledgements Authors would like to thank Mr Wen-Shao Yen for bacterial isolation. This research work was kindly supported by grants from the National International Journal of Systematic and Evolutionary Microbiology 63
Pseudomonas formosensis sp. nov. Science Council, Council of Agriculture, Executive Yuan and in part by the Ministry of Education, Taiwan, ROC. under the Aim for the Top University (ATU) plan.
References Ait Tayeb, L., Ageron, E., Grimont, F. & Grimont, P. A. D. (2005).
Molecular phylogeny of the genus Pseudomonas based on rpoB sequences and application for the identification of isolates. Res Microbiol 156, 763–773. 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. Collins, M. D. (1985). Isoprenoid quinone analysis in classification
and identification. In Chemical Methods in Bacterial Systematics, pp. 267–287. Edited by M. Goodfellow & D. E. Minnikin. London: Academic Press. Colwell, R. R. (1965). Proposal of a neotype, ATCC 14216, for
Pseudomonas aeruginosa (Schroeter 1872) Migula 1900 and request for an opinion. Int Bull Bacteriol Nomencl Taxon 15, 87–96. Delisle, F., Lesage, D., Gayral, J. P., Daguet, G. L., Vergez, P. & Le Cunff, D. (1986). [Evaluation of the antibiotic sensitivity of
Pseudomonas sp. using the API-ATB-PSE system. Comparison with diffusion and the reference agar dilution method]. Pathol Biol (Paris) 34, 424–429 (in French). Felsenstein, J. (1985). Confidence limits on phylogenies: an approach
using the bootstrap. Evolution 39, 783–791. GCG (1995). Wisconsin Package Version 8.1 Program Manual.
procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241. Moore, E. R. B., Mau, M., Arnscheidt, A., Bo¨ttger, E. C., Hutson, R. A., Collins, M. D., van de Peer, Y., de Wachter, R. & Timmis, K. N. (1996).
The determination and comparison of the 16S rRNA gene sequences of species of the genus Pseudomonas (sensu stricto) and estimation of the natural intrageneric relationships. Syst Appl Microbiol 19, 478–492. Murray, R. G. E., Doetsch, R. N. & Robinow, C. F. (1994).
Determinative and cytological light microscopy. In Methods for General and Molecular Bacteriology, pp. 12–41. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology. Oyaizu, H. & Komagata, K. (1983). Grouping of Pseudomonas species
on the basis of cellular fatty acid composition and the quinone system with special reference to the existence of 3-hydroxy fatty acids. J Gen Appl Microbiol 29, 17–40. Paisley, R. (1996). MIS Whole Cell Fatty Acid Analysis by Gas
Chromatography Training Manual. Newark, DE: MIDI. Palleroni, N. J. (1984). Genus I. Pseudomonas Migula 1984, 237AL. In
Bergey’s Manual of Systematic Bacteriology, vol. 1, pp. 141–199. Edited by N. R. Krieg & J. G. Holt. Baltimore: Williams & Wilkins. Palleroni, N. J. (1993). Pseudomonas classification. A new case history
in the taxonomy of Gram-negative bacteria. Antonie van Leeuwenhoek 64, 231–251. Pascual, J., Lucena, T., Ruvira, M. A., Giordano, A., Gambacorta, A., Garay, E., Arahal, D. R., Pujalte, M. J. & Macia´n, M. C. (2012).
Pseudomonas litoralis sp. nov., isolated from Mediterranean seawater. Int J Syst Evol Microbiol 62, 438–444.
Madison, WI: Genetics Computer Group.
Peix, A., Ramı´rez-Bahena, M. H. & Vela´zquez, E. (2009). Historical
Gibello, A., Vela, A. I., Martı´n, M., Mengs, G., Alonso, P. Z., Garbi, C. & Ferna´ndez-Garayza´bal, J. F. (2011). Pseudomonas composti sp. nov.,
evolution and current status of the taxonomy of genus Pseudomonas. Infect Genet Evol 9, 1132–1147.
isolated from compost samples. Int J Syst Evol Microbiol 61, 2962– 2966. Heiner, C. R., Hunkapiller, K. L., Chen, S. M., Glass, J. I. & Chen, E. Y. (1998). Sequencing multimegabase-template DNA with BigDye
Rossello´, R. A., Garcı´a-Valde´s, E., Lalucat, J. & Ursing, J. (1991).
Genotypic and phenotypic diversity of Pseudomonas stutzeri. Syst Appl Microbiol 14, 150–157. Sasser, M. (1990). Identification of bacteria by gas chromatography of
terminator chemistry. Genome Res 8, 557–561.
cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI Inc.
Hung, M.-H., Bhagwath, A. A., Shen, F.-T., Rekha, P. D. & Young, C.-C. (2005). Indigenous rhizobia associated with native shrubby
Seldin, L. & Dubnau, D. (1985). Deoxyribonucleic acid homology
legumes in Taiwan. Pedobiologia 49, 577–584. Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-e:
a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62, 716– 721.
among Bacillus polymyxa, Bacillus macerans, Bacillus azotofixans, and other nitrogen-fixing Bacillus strains. Int J Syst Bacteriol 35, 151– 154. Sneath, P. H. A., Stevens, M. & Sackin, M. J. (1981). Numerical
taxonomy of Pseudomonas based on published records of substrate utilization. Antonie van Leeuwenhoek 47, 423–448.
Lai, Q. & Shao, Z. (2008). Pseudomonas xiamenensis sp. nov., a
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using
denitrifying bacterium isolated from activated sludge. Int J Syst Evol Microbiol 58, 1911–1915.
maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739.
Maidak, B. L., Cole, J. R., Lilburn, T. G., Parker, C. T., Jr, Saxman, P. R., Farris, R. J., Garrity, G. M., Olsen, G. J., Schmidt, T. M. & Tiedje, J. M. (2001). The RDP-II (Ribosomal Database Project). Nucleic Acids Res
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible
29, 173–174. Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise
measurement of the G+C content of deoxyribonucleic acid by high performance liquid chromatography. Int J Syst Bacteriol 39, 159–167. ¨ ber ein neues System der Bakterien. Arb Bakteriol Migula, W. (1894). U Inst Karlsruhe 1, 235–238 (in German).
strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882. Vancanneyt, M., Segers, P., Torck, U., Hoste, B., Bernardet, J.-F., Vandamme, P. & Kersters, K. (1996). Reclassification of
Flavobacterium odoraturn (Stutzer 1929) strains to a new genus, Myroides, as Myroides odoratus comb. nov. and Myroides odoratimimus sp. nov. Int J Syst Bacteriol 46, 926–932.
Miller, L. T. (1982). Single derivatization method for routine analysis of bacterial whole-cell fatty acid methyl esters, including hydroxy acids. J Clin Microbiol 16, 584–586.
Watts, D. & MacBeath, J. R. (2001). Automated fluorescent DNA
Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, K. & Parlett, J. H. (1984). An integrated
Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. &
http://ijs.sgmjournals.org
sequencing on the ABI PRISM 310 Genetic Analyzer. Methods Mol Biol 167, 153–170.
3173
S.-Y. Lin and others other authors (1987). International Committee on Systematic
Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463– 464.
isolated from the sludge of an anaerobic ammonium-oxidizing bioreactor. Int J Syst Evol Microbiol 59, 2594–2598. Yamamoto, S. & Harayama, S. (1995). PCR amplification and direct
Xiao, Y.-P., Hui, W., Wang, Q., Roh, S. W., Shi, X.-Q., Shi, J.-H. & Quan, Z.-X. (2009). Pseudomonas caeni sp. nov., a denitrifying bacterium
sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains. Appl Environ Microbiol 61, 1104–1109.
3174
International Journal of Systematic and Evolutionary Microbiology 63
