Piscinibacterium candidicorallinum gen. nov., sp. nov., a member of [PDF]

International Journal of Systematic and Evolutionary Microbiology (2016), 66, 5260–5267

DOI 10.1099/ijsem.0.001505

Piscinibacterium candidicorallinum gen. nov., sp. nov., a member of the order Burkholderiales isolated from a fish pond Shih-Yi Sheu,1 Yi-Shu Li2 and Wen-Ming Chen2 Correspondence Wen-Ming Chen

1

Department of Marine Biotechnology, National Kaohsiung Marine University, No. 142, Hai-Chuan Rd, Nan-Tzu, Kaohsiung City 811, Taiwan, ROC

[email protected]

2

Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung Marine University, No. 142, Hai-Chuan Rd, Nan-Tzu, Kaohsiung City 811, Taiwan, ROC

A bacterial strain designated LYH-15T was isolated from a freshwater fish pond in Taiwan and characterized using a polyphasic taxonomy approach. Cells of LYH-15T were Gram-stainingnegative, aerobic, motile by means of a single polar flagellum, poly-b-hydroxybutyrate-containing, non-spore forming, straight rods and formed light-coral-colored colonies. Growth occurred at 15–40  C (optimum, 30  C), at pH 5.0–9.0 (optimum, pH 7.0) and with 0–0.5 % NaCl (optimum, 0 %). Phylogenetic analyses based on 16S rRNA gene sequences showed that LYH-15T forms a distinct phyletic line within the order Burkholderiales, with less than 94 % sequence similarity to its closest relatives with validly published names. The predominant fatty acids were summed feature 3 (comprising C16 : 1!7c and/or C16 : 1!6c), C16 : 0 and C18 : 1!7c. The major isoprenoid quinone was Q-8 and the DNA G+C content was 63.8 mol%. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and several uncharacterized lipids. The major polyamines were 2-hydroxyputrescine and putrescine. On the basis of the genotypic and phenotypic data, LYH-15T represents a novel species of a new genus in the order Burkholderiales, for which the name Piscinibacterium candidicorallinum gen. nov., sp. nov. is proposed. The type strain is LYH-15T (=BCRC 80969T=LMG 29480T=KCTC 52168T).

The order Burkholderiales, belonging to the class Betaproteobacteria, is a morphologically, metabolically and ecologically diverse group. Members of this order are strictly aerobic and facultatively anaerobic chemoorganotrophs, obligate and facultative chemolithotrophs, nitrogen-fixing organisms and plant, animal and human pathogens (Garrity et al., 2005). This order has five identified families based on 16S rRNA gene sequence analysis: Alcaligenaceae, Burkholderiaceae, Comamonadaceae, Oxalobacteraceae and Sutterellaceae (the LPSN website, List of Prokaryotic Names with Standing in Nomenclature; http://www.bacterio.net/burkholderiales.html), which were isolated from various habitats, such as freshwater, mudflow deposits, activated sludge biomass, soil, plant, cystic fibrosis patients, human faeces and ciliate. In addition, several genera such as

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Piscinibacterium candidicorallinum strain LYH-15T is LT158233. Three supplementary figures and one supplementary table are available with the online Supplementary Material.

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Aquabacterium, Aquincola, Ideonella, Inhella, Leptothrix, Methylibium, Mitsuaria, Paucibacter, Piscinibacter, Rivibacter, Rubrivivax, Sphaerotilus, Tepidimonas, Thiobacter, Thiomonas and Xylophilus are placed in incertae sedis (of uncertain position) as unclassified Burkholderiales (the LPSN website; http://www.bacterio.net/-classifphyla.html). During the characterization of micro-organisms from a freshwater sample collected from a fish pond in Sanyi Township, Miaoli County (GPS location: 22 54¢ 40† N 121 14¢ 13† E; pH 7.0, 25  C) in Taiwan, a bacterial strain, designated LYH-15T, was isolated on R2A agar (BD Difco), and subjected to detailed taxonomic analyses. Sub-cultivation was performed on R2A agar at 25  C for 48–72 h. The isolate was preserved at 80  C in R2A broth with 20 % (v/ v) glycerol or by lyophilization. Genomic DNA was isolated using a bacterial genomic kit (DP02-150, GeneMark Technology) and the 16S rRNA gene sequence was analyzed as described previously by Chen et al. (2001). Primers fD1 (5¢-AGAGTTTGATCC TGGCTCAG-3¢) and rD1 (5¢-AAGGAGGTGATCCAGCC3¢) were used for amplification of bacterial 16S rRNA

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Piscinibacterium candidicorallinum gen. nov., sp. nov.

genes by PCR. These primers correspond to nucleotide positions 8–27 and 1525–1541 of the Escherichia coli 16S rRNA gene, respectively, and can be used for amplifying nearly full-length 16S rRNA gene sequences. The PCR product was purified, and direct sequencing was performed by using sequencing primers fD1, rD1, 520F and 800R (Weisburg et al., 1991; Anzai et al., 1997) with a DNA sequencer (ABI Prism 3730; Applied Biosystems). The sequenced length of the 16S rRNA gene was 1415 bp for LYH-15T and this gene sequence was compared with those available from the EzTaxon-e (Kim et al., 2012), the Ribosomal Database Project (Cole et al., 2009) and GenBank databases (http://blast.ncbi.nlm.nih.gov/Blast.cgi.). Analysis of the sequence data was performed by using the software package BioEdit (Hall, 1999) and MEGA 7 (Kumar et al., 2016), after multiple alignments of the data by CLUSTAL_X (Thompson et al., 1997). Distances (corrected according to Kimura’s two-parameter model; Kimura, 1983) were calculated and clustering was performed with the neighbour-joining method (Saitou & Nei, 1987). The maximum-likelihood (Felsenstein, 1981), maximum-parsimony (Kluge & Farris, 1969) and minimum-evolution (Rzhetsky & Nei, 1993) trees were generated by using the treeing algorithms contained in the PHYLIP software package (Felsenstein, 1993). In each case bootstrap values were calculated based on 1000 replications. The phylogenetic analyses based on 16S rRNA gene sequences showed that LYH-15T formed a distinct phylogenetic lineage in the order Burkholderiales within the class Betaproteobacteria in the neighbour-joining tree (Fig. 1). The overall topologies of the maximum-likelihood and maximum-parsimony trees were similar. These findings were confirmed by analysis based on the minimum-evolution algorithm [Fig. S1, (available in the online Supplementary Material)]. In all phylogenetic trees, this distinct phylogenetic lineage could not be associated with any known families in the order Burkholderiales. Sequence similarity calculations (over 1400 bp) indicated that LYH-15T shared less than 94 % sequence similarity to its closest relatives with validly published names. LYH-15T was closely related to the species of the genera belonging to the unclassified Burkholderiales, such as Leptothrix (92.0–93.3 % sequence similarity), Methylibium (93.3 %), Rhizobacter (92.5–93.3 %), Paucibacter (93.1 %), Aquincola (92.9 %) and Piscinibacter (92.7 %), and the genera Pandoraea (91.7–93.3 %) in the family Burkholderiaceae, Kinneretia (93.1 %) in the family Comamonadaceae, Derxia (92.6– 93.0 %) in the family Alcaligenaceae, and Glaciimonas (91.6–92.9 %) and Herbaspirillum (91.9–92.8 %) in the family Oxalobacteraceae. The morphology of bacterial cells was observed by phasecontrast microscopy (model DM2000; Leica) and transmission electron microscopy (H-7500; Hitachi) (Fig. 2) using cells grown in R2A broth at 30  C for lag, exponential and stationary phases of growth. Cellular motility was tested by the hanging-drop method (Beveridge et al., 2007). The Gram Stain Set S (BD Difco) kit and the Ryu non-staining http://ijs.microbiologyresearch.org

KOH method (Powers, 1995) were used for testing the Gram reaction. The presence of a capsule was assessed using the Hiss staining method (Beveridge et al., 2007). Colony morphology was observed on R2A agar using a stereoscopic microscope (SMZ 800; Nikon). Poly-b-hydroxybutyrate granule accumulation was examined under light microscopy after staining of the cells with Sudan black (Schlegel et al., 1970) and visualized by UV illumination after directly staining growing bacteria on plates containing Nile red (Spiekermann et al., 1999). The pH range for growth was determined by measuring the OD600 of R2A broth. The pH was adjusted prior to sterilization to pH 4–9 (at intervals of 1.0 pH unit) using appropriate biological buffers (Breznak & Costilow, 2007): citrate– Na2HPO4 buffer, pH range 4.0–5.0; phosphate buffer, pH range 6.0–7.0; Tris buffer, pH range 8.0–9.0. Verification of the pH values after autoclaving revealed only minor changes. The temperature range for growth was determined in R2A broth at 4–50  C (4, 10, 15, 20, 25, 30, 35, 37, 40, 45 and 50  C). To investigate the tolerance to NaCl, R2A broth was prepared according to the formula of the BD Difco medium with NaCl concentration adjusted to 0, 0.5 and 1.0–6.0 %, w/v (at intervals of 1.0 %). Growth under anaerobic condition was determined after incubating LYH-15T on R2A agar in the Oxoid AnaeroGen system. Growth was tested on nutrient agar, trypticase soy agar, R2A agar and LB agar (all from Difco). LYH-15T was examined for a broad range of phenotypic properties. Activities of catalase, oxidase, DNase, urease and lipase (corn oil), hydrolysis of starch, casein, gelatin and Tweens 20, 40, 60 and 80 were determined using standard methods (Tindall et al., 2007). Chitin hydrolysis activity was determined using chitinase-detection agar plates (CDA plates). Chitin hydrolysis was visualized by the formation of a clear zone around the colonies on CDA plates. CDA plates were prepared as described earlier by Wen et al. (2002). Hydrolysis of carboxymethylcellulose (CM-cellulose) was tested as the method described by Bowman (2000) using R2A agar as the basal medium. Additional, biochemical tests were performed using API ZYM and API 20NE kits (both from bioMerieux) and carbon source utilization was evaluated using the GN2 microplate (Biolog). All commercial phenotypic tests were performed according to the manufacturers’ recommendations. Sensitivity to antibiotics of LYH-15T was tested by the disc diffusion method after spreading cell suspensions (0.5 McFarland) on R2A agar. The discs (Oxoid) contained the following antibiotics: ampicillin (10 µg), chloramphenicol (30 µg), gentamicin (10 µg), kanamycin (30 µg), nalidixic acid (30 µg), novobiocin (30 µg), rifampicin (5 µg), penicillin G (10 U), streptomycin (10 µg), sulfamethoxazole (23.75 µg) plus trimethoprim (1.25 µg) and tetracycline (30 µg). The effect of antibiotics on cell growth was assessed after 2 days at 30  C. The diameter of the antibiotic discs was 8 mm. The strain was considered susceptible when the diameter of the inhibition zone was >13 mm, intermediate

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S.-Y. Sheu, Y.-S. Li and W.-M. Chen

81 0.01 91 74

100 100

Rhodoferax fermentans FR2T (D16211)

100 99

96 81

Albidiferax ferrireducens T118T (CP000267) Variovorax paradoxus IAM 12373T (D88006) Curvibacter gracilis 7-1T (AB109889)

Ramlibacter tataouinensis TTB310T (CP000245)

Comamonadaceae

Malikia granosa P1T (AJ627188) Zhizhongheella caldifontis YIM 78140T (KF771277)

84 77 79

Caldimonas manganoxidans JCM 10698T (AB008801) Schlegelella thermodepolymerans K14T (AY152824) 82

Rivibacter subsaxonicus BF49T (AM774413) Methylibium petroleiphilum PM1T (CP000555) Piscinibacter aquaticus IMCC1728T (DQ664244) Aquabacterium commune B8T (AF035054)

96 94

Rubrivivax gelatinosus ATCC 17011T (D16213)

81

Aquincola tertiaricarbonis L10T (DQ656489)

96 94

95 92

89

Mitsuaria chitosanitabida 3001T (AB006851) T

Paucibacter toxinivorans 2C20 (AY515390)

Unclassified Burkholderiales

Sphaerotilus natans DSM 6575T (AZRA01000044)

80

Leptothrix discophora SS-1T (L33975) Thiomonas intermedia ATCC 15466T (AY455809) Piscinibacterium candidicorallinum LYH-15T (LT158233) Eoetvoesia caeni PB3-7BT (FJ948170) Pusillimonas noertemannii BN9T (AY695828) 95

Parapusillimonas granuli Ch07T (DQ466075) Candidimonas nitroreducens SC-089T (FN556191)

77 79

100 100

97 99

Bordetella pertussis Tohama IT (BX470248) Achromobacter xylosoxidans NBRC 15126T (CP006958)

Alcaligenaceae

Pigmentiphaga kullae K24T (AF282916) Derxia gummosa IAM 13946T (AB089482) Lautropia mirabilis ATCC 51599T (GL636064)

100 99

Pandoraea apista CCUG 38412T (AY268172) Burkholderia cepacia Ballard 717T (U96927)

Burkholderiaceae

T

Cupriavidus necator N-1 (AF191737) Noviherbaspirillum malthae CC-AFH3T (DQ490985) 92 90

Oxalicibacterium flavum TA17T (AY061962) 70 76

Glaciimonas immobilis Cr9-30T (GU441679) Oxalobacter formigenes OxBT (U49757)

Oxalobacteraceae

Paraherbaspirillum soli JS5-2T (FJ812351) Herbaspirillum seropedicae DSM 6445T (Y10146)

Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing relationships between Piscinibacterium candidicorallinum LYH-15T and closely related taxa within the order Burkholderiales. Numbers at nodes are bootstrap percentages >70 % based on the neighbour-joining (above nodes) and maximum-parsimony (below nodes) tree-making algorithms. Filled circles indicate branches of the tree that were also recovered using the maximum-likelihood and maximumparsimony tree-making algorithms. Open circles indicate that the corresponding nodes were also recovered in the tree generated with the maximum-parsimony algorithm. Bar, 0.01 substitutions per nucleotide position.

at 10–12 mm and resistant at 10 %) were summed feature 3 (comprising C16 : 1!7c and/or C16 : 1!6c), C16 : 0 and C18 : 1!7c.

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Piscinibacterium candidicorallinum gen. nov., sp. nov.

Burkholderiales. The DNA G+C content of LYH-15T, determined by HPLC according to the method of Mesbah et al. (1989), was 63.8±1.0 mol%.

Fig. 2. Transmission electron micrograph of cells of Piscinibacterium candidicorallinum LYH-15T and showing a single polar flagellum (arrow). Cells were grown on R2A broth for 48 h at 30  C. Bar, 0.5 µm.

Polar lipids were extracted and analyzed by two-dimensional TLC according to the protocol of Embley & Wait (1994). Molybdophosphoric acid was used for the detection of the total polar lipids, ninhydrin for amino lipids, the Zinzadze reagent for phospholipids, the Dragendorff reagent for choline-containing lipids and the a-naphthol reagent for glycolipids. LYH-15T exhibited a complex polar lipid profile consisting of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), two uncharacterized aminophospholipids (APL1 and APL2), four uncharacterized phospholipids (PL1–PL4), an uncharacterized glycolipid (GL) and an uncharacterized aminolipid (AL) (Fig. S2). Polyamines were extracted from LYH-15T, and analysis was carried out as described by Busse & Auling (1988) and Busse et al. (1997). Cells were cultivated in R2-PYE medium (l 1 : 0.75 g peptone from casein, 0.75 g yeast extract, 0.3 g K2 HPO4, 0.024 g MgSO4, pH 7.2) at 30  C for 3 days, and homogenized in 0.2 M perchloric acid (HClO4) and centrifuged. Polyamines in the resultant supernatant were analyzed by HPLC on D-7000 high-speed liquid chromatograph (Hitachi). The polyamine pattern of LYH-15Tcontained 2-hydroxyputrescine (HPUT, 50.8 %), putrescine (PUT, 48.2 %) and spermidine (SPD, 1.0 %) (Fig. S3). Isoprenoid quinones were extracted and purified according to the method of Collins (1994) and were analyzed by HPLC. LYH-15T had Q-8 as the major respiratory quinone, which is the same as most members of the order http://ijs.microbiologyresearch.org

Detailed results of physiological, biochemical and morphological characterization of LYH-15T are provided in the genus and species descriptions and Table 1. Several morphological, physiological and biochemical properties, such as colony pigmentation, cell shape, presence of cell motility, chemotropic and aerobic growth, inability to grow with higher NaCl concentrations (>0.5 %), presence of catalase activity and ability to reduce nitrate to nitrite, distinguish LYH-15T from all members of the phylogenetically related genera in the order Burkholderiales, such as Leptothrix, Methylibium, Paucibacter, Rhizobacter, Aquincola, Piscinibacter, Pandoraea, Kinneretia, Derxia, Glaciimonas and Herbaspirillum (Table 1). More importantly, LYH-15T could be also differentiated from members of the order Burkholderiales based on chemotaxonomic characteristics, such as the distinctive major fatty acids, major polar lipids and the DNA G+C content (Table 1). Additionally, the fatty acid profile of LYH-15Twas compared with those of members of the most phylogenetically closely related genera in Table S1. Summed feature 3 (comprising C16 : 1!7c and/or C16 : 1!6c) and C16 : 0 were commonly found as the major components in the type strains of phylogenetically related genera in the order Burkholderiales (Tables 1 and S1). However, Pandoraea apista LMG 16407T had significant amounts of cyclopropane acids such as C17 : 0 cyclo and C19 : 0 cyclo !8c, but these two fatty acids were not detected in LYH-15T. And, Paucibacter toxinivorans 2C20T and Aquincola tertiaricarbonis L10T contained C14 : 0, C15 : 0 and C17 : 0, but these three fatty acids were not found in LYH-15T. LYH-15T had a much higher proportion of fatty acid C18 : 1!7c than Piscinibacter aquaticus IMCC1728T. Furthermore, several hydroxyl fatty acids such as C8 : 0 3-OH, C12 : 0 2-OH, C12 : 0 3-OH, C14 : 0 2-OH, C14 : 0 3-OH, C16 : 0 2-OH and C16 : 0 3OH were detected in Methylibium petroleiphilum PM1T, Rhizobacter dauci H6T, Pandoraea apista LMG 16407T, Kinneretia asaccharophila KIN192T, Derxia gummosa LMG 3977T, Glaciimonas immobilis Cr9-30T or Herbaspirillum seropedicae IAM 14977T, but not detected in LYH-15T. It is now generally accepted that 16S rRNA gene sequence similarities between two bacteria of less than 95 % are an indication of affiliation to different genera (Ludwig et al., 1998). LYH-15T most probably represents a species of a new genus, since the 16S rRNA gene sequence similarity to its closest relatives with validly published names, Leptothrix discophora SS-1T, Pandoraea thiooxydans ATSB16T, Methylibium petroleiphilum PM1T and Rhizobacter profundi DS486-5T is only 93.3 %. Moreover, LYH-15T can be readily distinguished from these closest phylogenetic neighbours by chemotaxonomic, physiological and biochemical characteristics. Also, the low levels of 16S rRNA gene sequence similarity between LYH-15T and all other members of the order Burkholderiales together with differential phenotypic properties (Table 1), indicate that strain LYH-15T represents a novel species of a new genus within the order

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5264

+

Motility

ND

C16 : 0, SF3

C16 : 0, *SF3, C18 : 1!7c

PE, PG, DPG

Distinctive major fatty acids (>10 %)

Major polar lipids

ND

ND

+

ND

+

6–8.5

5–9

%0.5

Nitrate reduction

10–37

15–40

aerobic

+

straight rod

darkbrown

Sewage, freshwater, sediment

2

Maximum NaCl concentration (%, w/v) Catalase

Temperature range for growth ( C) pH range for growth

aerobic

straight rod

Cell shape

Facultatively chemolithotrophic Relation to O2

lightcoral

Fish pond

1

Colony pigmentation

Source

Characteristic

ND

C16 : 0, SF3c

+

ND

ND

37

facultatively anaerobic

+

straight rod

cream, yellow, brown

Biofilter

3

PE, PG, DPG

C16 : 0, SF3, C17 : 0 cyclo, C18 : 1!7c

V

V

%0.7

5–10

aerobic, facultatively anaerobic, microaerophilic 7–35

+

straight to slightly curved rod

white, yellow, brown

Soil, plant, sediment

4

ND

C16 : 0, SF3, C18 : 1!7c

ND

W

ND

ND

37

aerobic

+

rod

ND

Sediment

5

PE, PG

C16 : 0, SF3

W

ND

6–8

4–40

aerobic

+

rod

ND

Groundwater, wastewater, treatment plant

6

PE, PG, DPG

C16 : 0, SF3

1.0

6–10

15–42

facultatively anaerobic

short rod

beigemilkish

Freshwater pond

7

C16 : 0, SF3, C17 : 0 cyclo, C18 : 1!7c, C19 : 0 cyclo !8c PE, PG, DPG, PME

V

V

5.0

4–10

4–42

aerobic

+

+

straight rod

cream

Patient, soil, chicken dung

8

ND

C16 : 0, SF3, C18 : 1!7c

+

ND

ND

20–37

aerobic

+

rod

cream

Freshwater lake

9

PE, PG, DPG

C16 : 0, SF3, C18 : 1!7c

V

0–4

5.5–9

10–40

aerobic, microaerobic

+

rod

Light yellow, yellow

Soil, freshwater

10

PE, PG, DPG

C16 : 0, SF3, C17 : 0 cyclo, C18 : 1!7c

+

2.0

6–8

5–30

aerobic, microaerophilic

rod

white, pale cream

Alpine glacier, wastewater

11

ND

C16 : 0, SF3, C17 : 0 cyclo, C18 : 1!7c

V

+

3.0

5–8

4–40

aerobic, microaerobic

Well water, distilled water, soil, plant, fecal flora translucent, opaque, white, cream, yellowbrown, brown Slight curved rod, curved rod, spiral, vibrioid v

12

Taxa: 1, Piscinibacterium candidicorallinum gen. nov., sp. nov. (strain LYH-15); 2, Leptothrix (Spring et al., 1996); 3, Methylibium (Nakatsu et al., 2006); 4, Rhizobacter (Goto & Kuwata, 1988; Yoon et al., 2007; Stackebrandt et al., 2009; Imai et al., 2013; Wei et al., 2015; Jin et al., 2016); 5, Paucibacter (Rapala et al., 2005); 6, Aquincola (Lechner et al., 2007); 7, Piscinibacter (Song & Cho, 2007; Stackebrandt et al., 2009); 8, Pandoraea (Coenye et al., 2000; Anandham et al., 2010; Sahin et al., 2011); 9, Kinneretia (Gomila et al., 2010); 10, Derxia (Jensen et al., 1960; De smedt et al., 1980; Xie & Yokota, 2004; Chen et al., 2013); 11, Glaciimonas (Zhang et al., 2011; Chung et al., 2013; Frasson et al., 2015); 12, Herbaspirillum (Baldani et al., 1986, 1996; Kirchhof et al., 2001; Valverde et al., 2003; Ding & Yokota, 2004; Im et al., 2004; Rothballer et al., 2006; Jung et al., 2007; Dobritsa et al., 2010; Lagier et al., 2012). +, Positive reaction; W, weakly positive reaction; V, variable; , negative reaction; ND, no data available.

Table 1. Characteristics that differentiate LYH-15T from other phylogenetically related genera in the order Burkholderiales

S.-Y. Sheu, Y.-S. Li and W.-M. Chen

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*SF3, Summed feature 3 (C16 : 1!7c and/or C16 : 1!6c). PE, phosphatidylethanolamine; PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; PME, phosphatidylmethylethanolamine.

Q8

Burkholderiales, for which the name Piscinibacterium candidicorallinum gen. nov., sp. nov. is proposed.

57.9–67 49.1–55

Q8 Q8

69.2–72

ND

69.3

Q8

61.2–65.8

Q8

Q8

66.1–68

ND

Q8

66.6–70.8 69

Q8

68–71

Q8

63.8

Major ubiquinone

DNA G+C content (mol%)

Q8

69–70.5

66.2

10 3 Characteristic

Table 1. cont.

1

2

4

5

6

7

8

9

11

12

Piscinibacterium candidicorallinum gen. nov., sp. nov.

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Description of Piscinibacterium gen. nov. Piscinibacterium (Pi.sci.ni.bac.te¢ri.um. L. fem. n. piscina a pond; N.L. neut. n. bacterium, a small rod; N.L. neut. n. Piscinibacterium, a rod from a pond). Cells are Gram-staining-negative, aerobic, non-spore-forming, motile by means of a single polar flagellum and rodshaped. Oxidase and catalase are positive. The predominant quinone is Q-8. Major cellular fatty acids (>10 %) are summed feature 3 (comprising C16 : 1!7c and/or C16 : 1!6c), C16 : 0 and C18 : 1!7c. Phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, two uncharacterized aminophospholipids, four uncharacterized lipids, an uncharacterized glycolipid and an uncharacterized aminolipid are present in the polar lipid profile. The major polyamines are 2-hydroxyputrescine and putrescine. The type species is Piscinibacterium candidicorallinum.The DNA G+C content of the type strain of the type species is 63.8 mol%.

Description of Piscinibacterium candidicorallinum sp. nov. Piscinibacterium candidicorallinum (can.di.di.co.ral.li¢num. L. adj. candidus light-coloured; L. adj. corallinus coral-coloured; N.L. n. adj. candidicorallinum light-coral coloured). Displays the following properties in addition to those given in the genus description. Cells grow well on R2A agar and nutrient agar, but not on trypticase soy agar and LB agar. Cells are approximately 0.4–0.5 µm in diameter and 0.8–1.0 µm in length after 48 h of incubation on R2A agar at 30  C. Colonies are light-coral coloured, convex, round and smooth with entire edges. The colony size is approximately 0.5–0.7 mm in diameter on R2A agar after 48 h of incubation at 30  C. Growth occurs at 15–40  C (optimum, 30  C), at pH 5.0–9.0 (optimum, pH 7.0) and with 0–0.5 % NaCl (optimum, 0 %). Poly-b-hydroxybutyrate accumulation is observed. Positive for hydrolysis of starch, casein, CM-cellulose, corn oil and Tweens 20, 40, 60 and 80. Negative for hydrolysis of chitin and DNA. In API 20NE tests, positive reactions for nitrate reduction, aesculin and gelatin hydrolysis and assimilation of maltose, and negative reactions for indole production, glucose fermentation, arginine dihydrolase, urease and b-galactosidase activities, and assimilation of glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, gluconate, caprate, adipate, malate, citrate and phenylacetate are observed. In the API ZYM kit alkaline phosphatase, C4 esterase, C8 esterase lipase, C14 lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase and a-glucosidase activities are present and a-galactosidase, b-galactosidase, bglucuronidase, b-glucosidase, N-acetyl-b- glucosaminidase, a-mannosidase and a-fucosidase activities are absent. In the Downloaded from www.microbiologyresearch.org by IP: 192.3.100.152 On: Fri, 28 Dec 2018 21:45:37

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GN2 MicroPlate test, a-cyclodextrin, Tweens 40 and 80, Dmannose, pyruvic acid methyl ester, b-hydroxybutyric acid, D,L-lactic acid, L-alaninamide and L-proline are utilized as sole carbon sources, and dextrin, glycogen, a-D-glucose, sucrose, a-hydroxybutyric acid, a-ketobutyric acid, a-ketoglutaric acid and L-glutamic acid are weakly utilized as sole carbon sources. All other substrates in the GN2 microplate are not utilized. Sensitive to penicillin G, ampicillin, chloramphenicol, gentamicin, rifampicin, kanamycin, tetracycline, novobiocin, streptomycin, nalidixic acid and sulfamethoxazole plus trimethoprim. The major fatty acids (>10 %) are summed feature 3 (comprising C16 : 1!7c and/or C16 : 1!6c), C16 : 0 and C18 : 1!7c. The type strain is LYH-15T (=BCRC 80969T=LMG 29480T=KCTC 52168T) isolated from freshwater sample collected from a fish pond, in Sanyi Township, Miaoli County, Taiwan.

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