International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1036–1042
DOI 10.1099/ijs.0.057661-0
Undibacterium macrobrachii sp. nov., isolated from a freshwater shrimp culture pond Shih-Yi Sheu,1 Yang-Shun Lin,2 Jhen-Ci Chen1 and Wen-Ming Chen2 Correspondence Wen-Ming Chen [email protected]
1
Department of Marine Biotechnology, National Kaohsiung Marine University, No. 142, Hai-Chuan Rd, Nan-Tzu, Kaohsiung City 811, Taiwan, ROC
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 CMJ-9T was isolated from a freshwater shrimp culture pond in Taiwan and characterized using a polyphasic taxonomic approach. Cells of strain CMJ-9T were strictly aerobic, Gram-negative, motile by a single polar flagellum, poly-b-hydroxybutyratecontaining and formed light-yellow colonies. Growth occurred at 10–37 6C (optimum, 20– 30 6C), with 0–0.8 % NaCl (optimum, 0–0.1 %) and at pH 6.0–9.0 (optimum, pH 6.0–7.0). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain CMJ-9T belonged to the genus Undibacterium, and its closest neighbour was Undibacterium seohonense SHS5-24T, with 96.7 % sequence similarity. The predominant cellular fatty acids were summed feature 3 (C16 : 1v7c and/or C16 : 1v6c) and C16 : 0. The major cellular hydroxy fatty acid was C10 : 0 3-OH. The polar lipid profile consisted of the predominant lipids phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The polyamine profile was composed of the major compound putrescine and moderate amounts of 2-hydroxyputrescine. The major respiratory quinone was Q-8 and the DNA G+C content was 47.7 mol%. On the basis of the phylogenetic and phenotypic data, strain CMJ-9T should be classified within a novel species, for which the name Undibacterium macrobrachii sp. nov. is proposed. The type strain is CMJ-9T (5BCRC 80406T5LMG 26891T5KCTC 23916T).
The genus Undibacterium, proposed by Ka¨mpfer et al. (2007), belongs to the family Oxalobacteraceae of the order Burkholderiales in the class Betaproteobacteria (Garrity et al., 2005). At the time of writing, the genus Undibacterium comprised six species with validly published names: Undibacterium pigrum (type species; Ka¨mpfer et al., 2007), U. parvum (Ka¨mpfer et al., 2007; Eder et al., 2011), U. oligocarboniphilum (Eder et al., 2011), U. terreum (Liu et al., 2013), U. jejuense and U. seohonense (Kim et al., 2014). Cells are characteristically Gram-staining-negative, oxidase-positive, rod-shaped, motile by a single polar flagellum and chemoheterotrophic. Members of the genus possess Q-8 as the major respiratory quinone, phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol as predominant polar lipids, putrescine and 2-hydroxyputrescine as predominant polyamines, C16 : 0, summed feature 3 (C16 : 1v7c/C16 : 1v6c) and C18 : 1v7c as predominant fatty acids, C10 : 0 3-OH as the major hydroxy fatty acid and DNA G+C contents of 50.6–57.4 mol% (Ka¨mpfer et al., 2007; Eder et al., 2011; Liu et al., 2013). The present study was carried out to clarify the taxonomic The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CMJ-9T is JQ029110. A supplementary figure is available with the online version of this paper.
1036
position of the Undibacterium-like bacterial strain CMJ-9T by a polyphasic taxonomic approach. During the characterization of micro-organisms from a freshwater shrimp culture pond (GPS location 22u 349 410 N 120u 369 150 E; pH 8.5, 25 uC) in the Pingtung countryside of southern Taiwan, several bacterial colonies were isolated on R2A agar (BD Difco). Strains showing similar colony morphology were segregated and a representative strain, designated CMJ-9T, was selected for detailed analysis. Strain CMJ-9T was maintained on R2A agar at 25 uC. The strain was preserved at 280 uC as a 20 % (v/v) glycerol suspension in R2A broth (BD Difco) or by lyophilization with 20 % (w/v) skimmed milk. U. oligocarboniphilum DSM 21777T, U. parvum DSM 23061T and U. pigrum DSM 19792T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen. All three type strains were grown under the same conditions and used as reference strains for phenotypic and genotypic tests. Cells of strain CMJ-9T were observed by phase-contrast microscopy (DM 2000; Leica) in the lag, exponential and stationary phases of growth to ascertain the morphology, and colony morphology was examined using a stereoscopic microscope (SMZ 800; Nikon). Flagellar motility was tested using the hanging drop method, and the Spot Test 057661 G 2014 IUMS Printed in Great Britain
Undibacterium macrobrachii sp. nov.
flagella stain (BD Difco) was used for flagellum staining (Beveridge et al., 2007). The Gram reaction was examined by using the BD Difco Gram Stain set and according to the protocol of Powers (1995). Poly-b-hydroxybutyrate granule accumulation was examined as described by Schlegel et al. (1970) and Spiekermann et al. (1999). The pH range for growth was determined by measuring the OD600 of R2A broth cultures. The medium was adjusted prior to sterilization to pH 4–9 (at intervals of 1.0 pH unit) using appropriate biological buffers (Breznak & Costilow, 2007). To investigate the tolerance of NaCl, R2A broth was prepared according to the formula of the BD Difco medium with the NaCl concentration adjusted to 0–1.0 % (w/v) (at intervals of 0.1 %) and 2.0–5.0 % (at intervals of 1.0 %). The temperature range for growth was determined in R2A broth at 4–50 uC. Growth under anaerobic conditions was determined after incubating strain CMJ-9T in the Oxoid AnaeroGen system. Strain CMJ-9T and the three reference strains were examined for a broad range of phenotypic properties. Activities of catalase, oxidase, DNase and lipase (corn oil) and hydrolysis of starch, casein and Tweens 20, 40, 60 and 80 were determined according to standard methods (Tindall et al., 2007). Hydrolysis of CM-cellulose was tested as described by Bowman (2000) using R2A as the basal medium. Additional biochemical tests and carbon source utilization were investigated using the API ZYM, API 20NE and API 50CH kits (bioMe´rieux). Phenotypic tests using the API ZYM and API 50CH kits were performed according to the manufacturer’s recommendations. The 50CHB medium was used in the API 50CH tests. For the API 20NE (assimilation) tests, cells were suspended in diluted AUX medium (1 : 3 in deionized water) for strain CMJ-9T, U. oligocarboniphilum DSM 21777T and U. parvum DSM 23061T or AUX medium for U. pigrum DSM 19792T. Sensitivity of strain CMJ-9T to antibiotics was tested by the disc diffusion method after spreading cell suspensions (0.5 McFarland standard) on R2A agar plates. The discs (Oxoid) contained the following antibiotics: ampicillin (10 mg), chloramphenicol (30 mg), gentamicin (10 mg), kanamycin (30 mg), nalidixic acid (30 mg), novobiocin (30 mg), penicillin G (10 U), rifampicin (5 mg), streptomycin (10 mg), sulfamethoxazole plus trimethoprim (23.75/ 1.25 mg) and tetracycline (30 mg). The effect of antibiotics on cell growth was assessed after 3 days of incubation at 25 uC. The strains were considered susceptible or resistant as described by Nokhal & Schlegel (1983). Detailed results from phenotypic and biochemical analyses of strain CMJ9T are provided in Table 1 and in the species description. Genomic DNA was isolated by using a bacterial genomic kit and the 16S rRNA gene sequence was analysed as described previously by Chen et al. (2001). The sequenced length of the 16S rRNA gene was 1422 bp for strain CMJ9T, and this gene sequence was compared to those available from EzTaxon-e (Kim et al., 2012), the Ribosomal Database Project (Cole et al., 2009) and the GenBank database http://ijs.sgmjournals.org
(http://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequence analysis was performed by using the software package BioEdit (Hall, 1999) and MEGA version 5 (Tamura et al., 2011), 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). Maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Kluge & Farris, 1969) 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. 16S rRNA gene sequence analysis indicated that strain CMJ-9T belonged to the family Oxalobacteraceae of the order Burkholderiales in the class Betaproteobacteria. Phylogenetic analyses based on the 16S rRNA gene sequence revealed that strain CMJ-9T was closely related to the species of the genera Undibacterium (94.1–96.7 % sequence similarity), Herbaspirillum (94.3–95.4 %), Oxalicibacterium (93.9–95.2 %) and Herminiimonas (94.1–94.4 %). Strain CMJ-9T formed a distinct subline within the genus Undibacterium in the neighbour-joining tree (Fig. 1). The overall topologies of the maximum-likelihood and maximumparsimony trees were similar. Sequence similarity calculations (over 1400 bp) indicated that strain CMJ-9T was closely related to U. seohonense SHS5-24T (96.7 % 16S rRNA gene sequence similarity), U. oligocarboniphilum EM 1T (95.8 %), U. jejuense JS4-4T (95.6 %), U. pigrum CCUG 49009T (94.9 %), U. parvum CCUG 49012T (94.6 %) and U. terreum C3T (94.1 %). Representative members of all other genera listed in Fig. 1 showed lower sequence similarity (,95.4 %). The DNA G+C content of strain CMJ-9T, as determined by HPLC according to Mesbah et al. (1989), was 47.7± 1.0 mol%. Isoprenoid quinones were extracted and purified according to the method of Collins (1994) and were analysed by HPLC. Strain CMJ-9T had Q-8 as the major respiratory quinone, which is the same as that of all other species of the genus Undibacterium (Ka¨mpfer et al., 2007; Eder et al., 2011; Liu et al., 2013; Kim et al., 2014). The fatty acid profiles of strain CMJ-9T, U. oligocarboniphilum DSM 21777T, U. parvum DSM 23061T and U. pigrum DSM 19792T were analysed from cells grown on R2A agar at 25 uC for 3 days. The physiological age of the different bacterial cultures at the time of harvest was standardized by choice of sector from a quadrant streak on the R2A agar plates according to the MIDI protocol (http://www. microbialid.com/PDF/TechNote_101.pdf). In this study, the different type strains of the genus Undibacterium exhibited very similar growth rates on R2A agar. Fatty acid methyl esters were prepared and separated according to the instructions of the Microbial Identification System (MIDI; Sasser, 1990) and identified by MIDI version 6.0 and the RTSBA6.00 database. Strain CMJ-9T had a fatty acid profile similar to those of all other strains of Undibacterium examined (Table 2). The predominant cellular fatty acids 1037
S.-Y. Sheu and others
Table 1. Differential characteristics of strain CMJ-9T and type strains of species of the genus Undibacterium Strains: 1, CMJ-9T; 2, U. oligocarboniphilum DSM 21777T; 3, U. parvum DSM 23061T; 4, U. pigrum DSM 19792T; 5, U. terreum C3T (data from Liu et al., 2013); 6, U. jejuense JS4-4T (Kim et al., 2014); 7, U. seohonense SHS5-24T (Kim et al., 2014). Data were obtained in this study unless indicated. +, Positive; 2, negative; W, weakly positive reaction; ND, no data available. All strains are positive for assimilation of glucose and activities of alkaline phosphatase, leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase. All strains are negative for Gram staining, indole production, assimilation of mannitol, citrate and phenylacetate and activities of C14 lipase, a-galactosidase, b-galactosidase, b-glucuronidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase. Characteristic Isolation source Colony pigmentation on R2A agar Temperature range for growth (uC) NaCl range for growth (%) Hydrolysis of: Starch Tween 80 Enzyme activities C4 esterase C8 esterase lipase Valine arylamidase Cystine arylamidase Trypsin a-Chymotrypsin a-Glucosidase Biochemical characteristics (API 20NE) Nitrate reduction Glucose acidification Arginine dihydrolase activity Urease activity Aesculin hydrolysis Gelatin hydrolysis Assimilation of (API 20NE): Arabinose Mannose N-Acetylglucosamine Maltose Gluconate Caprate Adipate Malate Acid production from (API 50CH): Ribose Galactose Fructose Cellobiose Sucrose Trehalose Gentiobiose D-Fucose DNA G+C content (mol%)
1
2
3
4
5
6
7
Freshwater pond Light yellow 10–37 0–0.8
Purified water
Drinking water
Drinking water
Soil
Soil
Freshwater
Creamy 15–30 (10–35)* 0–0.5
Yellowish 10–25 (4–30)D 0–0.5
Beige 15–35 (4–30)D 0–1.0
Creamy 15–37 0–0.8
Creamy 10–33 0–0.5
Creamy 10–30 0
2 +
+ + (2)*
2 2
+ +
2 +
2 +
2 +
+ + + + + + 2
+ + + + 2 2 +
+ + 2 2 2 2 2
+ + + 2 2 2 +
+ + + + + + 2
+ + 2 2 2 2 2
2 2 + + 2 2 2
+ 2 2 2 2 2
+ 2 2 2 2 2 (W)*
+ 2 2 2 2 2
+ 2 2 2 + +
2 + + 2 2
2 2 2 2 + 2
+ 2 2 2 2 +
2 2 + + 2 2 + +
2 2 2 + + 2 2 2
2 2 2 + 2 2 2 2
2 + + + 2 2 2 2
+ + 2 2 + + + 2
2 + 2 + 2 2 2 2
2 2 2 + 2 2 2 2
2 2 2 + + 2 2 2 47.7
+ 2 2 2 + 2 2 2 (52.4)*
+ 2 2 2 2 2 2 2 (50.6)D
+ 2 + + + + + 2 (52.3)D
2 + + + 2 2 2 + 57.4
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
W
*Results in parentheses obtained from Eder et al. (2011) DResults in parentheses obtained from Ka¨mpfer et al. (2007).
(.10 %) of strain CMJ-9T were summed feature 3 (C16 : 1v7c and/or C16 : 1v6c; 52.9 %) and C16 : 0 (28.1 %). The major cellular hydroxy fatty acid was C10 : 0 3-OH
(8.8 %). The dominant fatty acids were summed feature 3 and C16 : 0 for the novel strain and all other type strains of the genus Undibacterium, which is consistent with previous
1038
International Journal of Systematic and Evolutionary Microbiology 64
Undibacterium macrobrachii sp. nov.
Collimonas fungivorans Ter6T (AJ310394) 97 ● 100 ● 97 Collimonas pratensis Ter91T (AY281137) 98 Collimonas arenae NCCB 100031T (AY281146) Glaciimonas immobilis Cr9-30T (GU441679)
0.01 ●
74
●
Herbaspirillum autotrophicum IAM 14942T (AB074524) Oxalicibacterium solurbis MY14T (AB008503) Herminiimonas aquatilis CCUG 36956T (AM085762)
96● 98 83
Herminiimonas fonticola S-94T (AY676462) Herminiimonas arsenicoxydans ULPAs1T (CU207211)
100● Herminiimonas saxobsidens NS11T (AM493906) 97 Herminiimonas glaciei UMB49T (EU489741) ● ●
Undibacterium terreum C3T (JQ417431) Undibacterium jejuense JS4-4T (KC735150) Undibacterium parvum CCUG 49012T (AM397629)
94● 90
Undibacterium macrobrachii CMJ-9T (JQ029110) Undibacterium seohonense SHS5-24T (KC735151)
●
●
Undibacterium oligocarboniphilum EM1T (GQ379228)
●
Undibacterium pigrum CCUG 49009T (AM397630) Massilia plicata 76T (AY966000) Paucimonas lemoignei LMG 2207T (X92555) Actimicrobium antarcticum KOPRI 25157T (HQ699437)
Oxalicibacterium flavum TA17T (AY061962) 92
Oxalicibacterium faecigallinarum YOxT (AB469788) Oxalicibacterium horti OD1T (AB469786) Burkholderia acidipaludis NBRC 101816T (AB513180)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of strain CMJ-9T and related taxa in the class Betaproteobacteria. Numbers at nodes are bootstrap percentages (.70 %) based on the neighbourjoining (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 maximum-parsimony tree-making algorithms; open circles indicate that the corresponding nodes were also recovered in the tree generated with the maximum-parsimony algorithm. Burkholderia acidipaludis NBRC 101816T was used as an outgroup. Bar, 0.01 substitutions per nucleotide position.
publications (Ka¨mpfer et al., 2007; Eder et al., 2011; Liu et al., 2013; Kim et al., 2014). Furthermore, the fatty acids C12 : 0 2-OH and C17 : 0 cyclo were detected in U. terreum C3T (Liu et al., 2013) but not in strain CMJ-9T or the other type strains of Undibacterium. In contrast to strain CMJ-9T and the other type strains of the genus Undibacterium, U. terreum C3T had much higher proportions of fatty acids C10 : 0 3-OH and C12 : 0 and a much lower proportion of summed feature 3. The polar lipids of strain CMJ-9T were extracted and analysed by two-dimensional TLC according to Embley & Wait (1994). Molybdophosphoric acid was used for the detection of total polar lipids, ninhydrin for amino lipids, Zinzadze reagent for phospholipids, Dragendorff reagent for choline-containing lipids and a-naphthol reagent for glycolipids. Strain CMJ-9T exhibited a complex polar lipid profile consisting of phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylserine, two uncharacterized aminolipids and http://ijs.sgmjournals.org
three uncharacterized polar lipids (Fig. S1, available in the online Supplementary Material). The predominant polar lipids were PE, PG and DPG; the possession of PE, PG and DPG as predominant polar lipids is consistent with previous descriptions of species of Undibacterium (Ka¨mpfer et al., 2007; Eder et al., 2011; Liu et al., 2013; Kim et al., 2014). Furthermore, strain CMJ-9T differed from the other type strains of the genus Undibacterium in the presence and proportions of some minor polar lipids. Polyamines were extracted from strain CMJ-9T and analysis was carried out as described by Busse & Auling (1988) and Busse et al. (1997). Cells were homogenized in 0.2 M perchloric acid (HClO4) and centrifuged. Polyamines in the resultant supernatant were treated with dansyl chloride solution (7.5 mg ml21 in acetone) and then analysed by HPLC on a Hitachi D-7000 chromatograph and UV-Vis detector L-7420 (Hitachi). The polyamine pattern of strain CMJ-9T contained putrescine (79.9 %), 2-hydroxyputrescine 1039
S.-Y. Sheu and others
Table 2. Cellular fatty acid compositions of strain CMJ-9T and type strains of species of the genus Undibacterium Strains: 1, CMJ-9T; 2, U. oligocarboniphilum DSM 21777T; 3, U. parvum DSM 23061T; 4, U. pigrum DSM 19792T; 5, U. terreum C3T (data from Liu et al., 2013); 6, U. jejuense JS4-4T (Kim et al., 2014); 7, U. seohonense SHS5-24T (Kim et al., 2014). All data were obtained in this study with cells grown on R2A agar at 25 uC for 3 days unless indicated. Values are percentages of total fatty acids; 2, ,1.0 % or not reported. Fatty acids that make up ,1.0 % of the total in all strains are not shown. Data from Eder et al. (2011) or Ka¨mpfer et al. (2007) are given in parentheses. For unsaturated fatty acids, the position of the double bond is located by counting from the methyl (v) end of the carbon chain; cis isomers are indicated by the suffix c. Fatty acid C10 : 0 C10 : 0 3-OH C12 : 0 C12 : 0 2-OH C14 : 0 C16 : 0 C17 : 0 cyclo C18 : 0 C18 : 1v7c 11-Methyl C18 : 1v7c Summed feature 3*
1
2
3
4
5
6
7
2 8.8 5.1 2 2 28.1 2 1.7 2.9 2 52.9
1.9 (0.5) 3.5 (2.3) 3.3 (2.9) 2 (2) 1.1 (0.4) 30.7 (33.5) 2 (2) 2.3 (2) 6.7 (7.7) 2 (1.3) 49.6 (51.1)
2 (3.0) 3.6 (5.2) 3.0 (3.2) 2 (2) 2 (0.8) 30.0 (23.2) 2 (2) 2.6 (2) 10.6 (2.1) 2 (2) 49.6 (62.5)
1.1 (1.0) 3.0 (4.7) 5.2 (5.3) 2 (2) 2.1 (1.5) 25.9 (22.6) 2 (2) 1.3 (2) 10.3 (9.0) 2 (2) 50.1 (55.7)
1.7 12.9 10.2 6.9 2 25.4 1.3 2 4.3 2 36.7
2 3.2 3.2 2 2 28.3 2 2 7.9 2 50.1
2 6.3 3.2 2 2 24.5 2 2 5.4 2 58.7
*Summed features are groups of two or three fatty acids that cannot be separated by GLC using the MIDI system. Summed feature 3 comprises C16 : 1v7c and/or C16 : 1v6c.
(12.4 %), spermidine (3.5 %), homospermidine (3.0 %) and spermine (1.2 %). Putrescine and 2-hydroxyputrescine were the major polyamines of strain CMJ-9T, in line with all other species of the genus Undibacterium for which the polyamine composition has been analysed (Ka¨mpfer et al., 2007; Eder et al., 2011; Liu et al., 2013). The physiological, biochemical and morphological characteristics of strain CMJ-9T are given in the species description and Table 1. Phenotypic examination revealed many common traits between the novel strain and the other species of the genus Undibacterium. However, strain CMJ-9T could be clearly differentiated from U. oligocarboniphilum DSM 21777T, U. parvum DSM 23061T, U. pigrum DSM 19792T, U. terreum C3T, U. jejuense JS4-4T and U. seohonense SHS5-24T by its colony pigmentation and by some enzyme activities and biochemical characteristics (Table 1).
Description of Undibacterium macrobrachii sp. nov. Undibacterium macrobrachii (ma.cro.bra9chi.i. N.L. n. Macrobrachium the scientific name of a genus of shrimp; N.L. gen. n. macrobrachii of Macrobrachium, referring to the isolation of the type strain from freshwater around shrimp belonging to the genus Macrobrachium).
Cells of strain CMJ-9T were Gram-staining-negative, oxidase-positive, motile by a single polar flagellum and chemoheterotrophic, growing under mesophilic and neutrophilic conditions. The quinone system, polyamine pattern, major characteristics of the polar lipid and fatty acid profiles and 16S rRNA gene sequence analysis suggested the placement of strain CMJ-9T in the genus Undibacterium. The phylogenetic inference is supported by the unique combination of chemotaxonomic and biochemical characteristics of the novel strain. Hence, strain CMJ-9T constitutes a member of the genus Undibacterium. The name Undibacterium macrobrachii sp. nov. is proposed for this taxon.
Cells are strictly aerobic, Gram-negative, flagellated and rod-shaped. Poly-b-hydroxybutyrate accumulation is observed. After 72 h of incubation on R2A agar at 25 uC, the mean cell size is 0.3–0.561.4–2.0 mm. Colonies on R2A agar are light yellow, circular, smooth and convex with entire edges. Colonies are approximately 1.0–1.3 mm in diameter on R2A agar after 72 h of incubation at 25 uC. Growth occurs at 10–37 uC, with 0–0.8 % NaCl and at pH 6.0–9.0. Optimum growth occurs at 20–30 uC, with 0–0.1 % NaCl and at pH 6.0–7.0. Positive for oxidase activity and hydrolysis of Tweens 20 and 80. Negative for catalase, DNase and lipase (corn oil) activities and hydrolysis of casein, starch, CM-cellulose and Tweens 40 and 60. In API 20NE tests, positive for nitrate reduction and assimilation of glucose, N-acetylglucosamine, maltose, adipate and malate and negative for indole production, fermentation of D-glucose, activities of arginine dihydrolase, urease and b-galactosidase, hydrolysis of aesculin and gelatin and assimilation of arabinose, mannose, mannitol, gluconate, caprate, citrate and phenylacetate. In API ZYM tests, alkaline phosphatase, C4 esterase, C8 esterase lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, acid phosphatase and
1040
International Journal of Systematic and Evolutionary Microbiology 64
Undibacterium macrobrachii sp. nov.
naphthol-AS-BI-phosphohydrolase activities are present; C14 lipase, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase activities are absent. The following compounds are utilized in the API 50CH test system: glucose, N-acetylglucosamine, cellobiose, maltose, sucrose, starch and glycogen. Negative for assimilation of glycerol, erythritol, D- and L-arabinose, ribose, D- and L-xylose, adonitol, methyl b-xyloside, galactose, fructose, mannose, sorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, methyl a-D-mannoside, methyl a-glucoside, amygdalin, arbutin, aesculin, salicin, lactose, melibiose, trehalose, inulin, melezitose, raffinose, xylitol, gentiobiose, turanose, lyxose, tagatose, D- and L-fucose, D- and L-arabitol, gluconate, 2-ketogluconate and 5ketogluconate. Sensitive to tetracycline, sulfamethoxazole plus trimethoprim, rifampicin, novobiocin, streptomycin, chloramphenicol, nalidixic acid, penicillin G, ampicillin, gentamicin and kanamycin. The predominant cellular fatty acids are summed feature 3 (C16 : 1v7c and/or C16 : 1v6c) and C16 : 0. The major cellular hydroxy fatty acid is C10 : 0 3-OH. The polar lipid profile consists of the predominant lipids phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The polyamine profile is composed of the major compound putrescine and moderate amounts of 2-hydroxyputrescine. The major respiratory quinone is Q-8. The type strain is CMJ-9T (5BCRC 80406T5LMG 26891T 5KCTC 23916T), isolated from a freshwater shrimp culture pond in the Pingtung countryside of southern Taiwan. The DNA G+C content of the type strain is 47.7 mol%.
Cole, J. R., Wang, Q., Cardenas, E., Fish, J., Chai, B., Farris, R. J., Kulam-Syed-Mohideen, A. S., McGarrell, D. M., Marsh, T. & other authors (2009). The Ribosomal Database Project: improved
alignments and new tools for rRNA analysis. Nucleic Acids Res 37, D141–D145. Collins, M. D. (1994). Isoprenoid quinones. In Chemical Methods in
Prokaryotic Systematics, pp. 265–309. Edited by M. Goodfellow & A. G. O’Donnell. Chichester: Wiley. Eder, W., Wanner, G., Ludwig, W., Busse, H.-J., Ziemke-Ka¨geler, F. & Lang, E. (2011). Description of Undibacterium oligocarboniphilum
sp. nov., isolated from purified water, and Undibacterium pigrum strain CCUG 49012 as the type strain of Undibacterium parvum sp. nov., and emended descriptions of the genus Undibacterium and the species Undibacterium pigrum. Int J Syst Evol Microbiol 61, 384–391. Embley, T. M. & Wait, R. (1994). Structural lipids of eubacteria. In Chemical Methods in Prokaryotic Systematics, pp. 121–161. Edited by M. Goodfellow & A. G. O’Donnell. Chichester: Wiley. Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a
maximum likelihood approach. J Mol Evol 17, 368–376. PHYLIP (phylogeny inference package), version 3.5c. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle, USA.
Felsenstein, J. (1993).
Garrity, G. M., Bell, J. A. & Lilburn, T. (2005). Family II. Oxalobac-
teraceae fam. nov. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2C, p. 623. Edited by D. J. Brenner, N. R. Krieg, J. T. Staley & G. M. Garrity. New York: Springer. Hall, T. A. (1999). BioEdit: a user-friendly biological sequence align-
ment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41, 95–98. Ka¨mpfer, P., Rossello´-Mora, R., Hermansson, M., Persson, F., Huber, B., Falsen, E. & Busse, H.-J. (2007). Undibacterium pigrum
gen. nov., sp. nov., isolated from drinking water. Int J Syst Evol Microbiol 57, 1510–1515. 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
References Beveridge, T. J., Lawrence, J. R. & Murray, R. G. E. (2007). Sampling
EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62, 716–721.
and staining for light microscopy. In Methods for General and Molecular Bacteriology, 3rd edn, pp. 19–33. Edited by T. J. Beveridge, J. A. Breznak, G. A. Marzluf, T. M. Schmidt & L. R. Snyder. Washington, DC: American Society for Microbiology.
Kim, S. J., Moon, J. Y., Weon, H. Y., Hong, S. B., Seok, S. J. & Kwon, S. W. (2014). Undibacterium jejuense sp. nov. and Undibacterium
Bowman, J. P. (2000). Description of Cellulophaga algicola sp. nov.,
Kimura, M. (1983). The Neutral Theory of Molecular Evolution. Cambridge: Cambridge University Press.
isolated from the surfaces of Antarctic algae, and reclassification of Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Cellulophaga uliginosa comb. nov. Int J Syst Evol Microbiol 50, 1861–1868. Breznak, J. A. & Costilow, R. N. (2007). Physicochemical factors in
growth. In Methods for General and Molecular Bacteriology, 3rd edn, pp. 309–329. Edited by T. J. Beveridge, J. A. Breznak, G. A. Marzluf, T. M. Schmidt & L. R. Snyder. Washington, DC: American Society for Microbiology. Busse, H.-J. & Auling, G. (1988). Polyamine pattern as chemotaxo-
nomic marker within the Proteobacteria. Syst Appl Microbiol 11, 1–8.
seohonense sp. nov., isolated from soil and freshwater, respectively. Int J Syst Evol Microbiol (in press).
Kluge, A. G. & Farris, F. S. (1969). Quantitative phyletics and the
evolution of anurans. Syst Zool 18, 1–32. Liu, Y. Q., Wang, B. J., Zhou, N. & Liu, S. J. (2013). Undibacterium
terreum sp. nov., isolated from permafrost soil. Int J Syst Evol Microbiol 63, 2296–2300. Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise
measurement of the G+C content of deoxyribonucleic acid by highperformance liquid chromatography. Int J Syst Bacteriol 39, 159–167. Nokhal, T. H. & Schlegel, H. G. (1983). Taxonomic study of
Busse, H.-J., Bunka, S., Hensel, A. & Lubitz, W. (1997). Discrimina-
Paracoccus denitrificans. Int J Syst Bacteriol 33, 26–37.
tion of members of the family Pasteurellaceae based on polyamine patterns. Int J Syst Bacteriol 47, 698–708.
Powers, E. M. (1995). Efficacy of the Ryu nonstaining KOH technique
for rapidly determining gram reactions of food-borne and waterborne bacteria and yeasts. Appl Environ Microbiol 61, 3756–3758.
Chen, W. M., Laevens, S., Lee, T. M., Coenye, T., De Vos, P., Mergeay, M. & Vandamme, P. (2001). Ralstonia taiwanensis sp. nov., isolated
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a
from root nodules of Mimosa species and sputum of a cystic fibrosis patient. Int J Syst Evol Microbiol 51, 1729–1735.
new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.
http://ijs.sgmjournals.org
1041
S.-Y. Sheu and others Sasser, M. (1990). Identification of bacteria by gas chromatography of
cellular fatty acids, Technical Note no. 101. Newark, DE: MIDI Inc.
maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739.
Schlegel, H. G., Lafferty, R. & Krauss, I. (1970). The isolation of mutants not accumulating poly-b-hydroxybutyric acid. Arch Mikrobiol
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible
71, 283–294.
strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Spiekermann, P., Rehm, B. H. A., Kalscheuer, R., Baumeister, D. & Steinbu¨chel, A. (1999). A sensitive, viable-colony staining method
using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Arch Microbiol 171, 73–80.
Tindall, B. J., Sikorski, J., Smibert, R. A. & Krieg, N. R. (2007).
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using
Phenotypic characterization and the principles of comparative systematics. In Methods for General and Molecular Bacteriology, 3rd edn, pp. 330–393. Edited by T. J. Beveridge, J. A. Breznak, G. A. Marzluf, T. M. Schmidt & L. R. Snyder. Washington, DC: American Society for Microbiology.
1042
International Journal of Systematic and Evolutionary Microbiology 64
DOI 10.1099/ijs.0.057661-0
Undibacterium macrobrachii sp. nov., isolated from a freshwater shrimp culture pond Shih-Yi Sheu,1 Yang-Shun Lin,2 Jhen-Ci Chen1 and Wen-Ming Chen2 Correspondence Wen-Ming Chen [email protected]
1
Department of Marine Biotechnology, National Kaohsiung Marine University, No. 142, Hai-Chuan Rd, Nan-Tzu, Kaohsiung City 811, Taiwan, ROC
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 CMJ-9T was isolated from a freshwater shrimp culture pond in Taiwan and characterized using a polyphasic taxonomic approach. Cells of strain CMJ-9T were strictly aerobic, Gram-negative, motile by a single polar flagellum, poly-b-hydroxybutyratecontaining and formed light-yellow colonies. Growth occurred at 10–37 6C (optimum, 20– 30 6C), with 0–0.8 % NaCl (optimum, 0–0.1 %) and at pH 6.0–9.0 (optimum, pH 6.0–7.0). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain CMJ-9T belonged to the genus Undibacterium, and its closest neighbour was Undibacterium seohonense SHS5-24T, with 96.7 % sequence similarity. The predominant cellular fatty acids were summed feature 3 (C16 : 1v7c and/or C16 : 1v6c) and C16 : 0. The major cellular hydroxy fatty acid was C10 : 0 3-OH. The polar lipid profile consisted of the predominant lipids phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The polyamine profile was composed of the major compound putrescine and moderate amounts of 2-hydroxyputrescine. The major respiratory quinone was Q-8 and the DNA G+C content was 47.7 mol%. On the basis of the phylogenetic and phenotypic data, strain CMJ-9T should be classified within a novel species, for which the name Undibacterium macrobrachii sp. nov. is proposed. The type strain is CMJ-9T (5BCRC 80406T5LMG 26891T5KCTC 23916T).
The genus Undibacterium, proposed by Ka¨mpfer et al. (2007), belongs to the family Oxalobacteraceae of the order Burkholderiales in the class Betaproteobacteria (Garrity et al., 2005). At the time of writing, the genus Undibacterium comprised six species with validly published names: Undibacterium pigrum (type species; Ka¨mpfer et al., 2007), U. parvum (Ka¨mpfer et al., 2007; Eder et al., 2011), U. oligocarboniphilum (Eder et al., 2011), U. terreum (Liu et al., 2013), U. jejuense and U. seohonense (Kim et al., 2014). Cells are characteristically Gram-staining-negative, oxidase-positive, rod-shaped, motile by a single polar flagellum and chemoheterotrophic. Members of the genus possess Q-8 as the major respiratory quinone, phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol as predominant polar lipids, putrescine and 2-hydroxyputrescine as predominant polyamines, C16 : 0, summed feature 3 (C16 : 1v7c/C16 : 1v6c) and C18 : 1v7c as predominant fatty acids, C10 : 0 3-OH as the major hydroxy fatty acid and DNA G+C contents of 50.6–57.4 mol% (Ka¨mpfer et al., 2007; Eder et al., 2011; Liu et al., 2013). The present study was carried out to clarify the taxonomic The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CMJ-9T is JQ029110. A supplementary figure is available with the online version of this paper.
1036
position of the Undibacterium-like bacterial strain CMJ-9T by a polyphasic taxonomic approach. During the characterization of micro-organisms from a freshwater shrimp culture pond (GPS location 22u 349 410 N 120u 369 150 E; pH 8.5, 25 uC) in the Pingtung countryside of southern Taiwan, several bacterial colonies were isolated on R2A agar (BD Difco). Strains showing similar colony morphology were segregated and a representative strain, designated CMJ-9T, was selected for detailed analysis. Strain CMJ-9T was maintained on R2A agar at 25 uC. The strain was preserved at 280 uC as a 20 % (v/v) glycerol suspension in R2A broth (BD Difco) or by lyophilization with 20 % (w/v) skimmed milk. U. oligocarboniphilum DSM 21777T, U. parvum DSM 23061T and U. pigrum DSM 19792T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen. All three type strains were grown under the same conditions and used as reference strains for phenotypic and genotypic tests. Cells of strain CMJ-9T were observed by phase-contrast microscopy (DM 2000; Leica) in the lag, exponential and stationary phases of growth to ascertain the morphology, and colony morphology was examined using a stereoscopic microscope (SMZ 800; Nikon). Flagellar motility was tested using the hanging drop method, and the Spot Test 057661 G 2014 IUMS Printed in Great Britain
Undibacterium macrobrachii sp. nov.
flagella stain (BD Difco) was used for flagellum staining (Beveridge et al., 2007). The Gram reaction was examined by using the BD Difco Gram Stain set and according to the protocol of Powers (1995). Poly-b-hydroxybutyrate granule accumulation was examined as described by Schlegel et al. (1970) and Spiekermann et al. (1999). The pH range for growth was determined by measuring the OD600 of R2A broth cultures. The medium was adjusted prior to sterilization to pH 4–9 (at intervals of 1.0 pH unit) using appropriate biological buffers (Breznak & Costilow, 2007). To investigate the tolerance of NaCl, R2A broth was prepared according to the formula of the BD Difco medium with the NaCl concentration adjusted to 0–1.0 % (w/v) (at intervals of 0.1 %) and 2.0–5.0 % (at intervals of 1.0 %). The temperature range for growth was determined in R2A broth at 4–50 uC. Growth under anaerobic conditions was determined after incubating strain CMJ-9T in the Oxoid AnaeroGen system. Strain CMJ-9T and the three reference strains were examined for a broad range of phenotypic properties. Activities of catalase, oxidase, DNase and lipase (corn oil) and hydrolysis of starch, casein and Tweens 20, 40, 60 and 80 were determined according to standard methods (Tindall et al., 2007). Hydrolysis of CM-cellulose was tested as described by Bowman (2000) using R2A as the basal medium. Additional biochemical tests and carbon source utilization were investigated using the API ZYM, API 20NE and API 50CH kits (bioMe´rieux). Phenotypic tests using the API ZYM and API 50CH kits were performed according to the manufacturer’s recommendations. The 50CHB medium was used in the API 50CH tests. For the API 20NE (assimilation) tests, cells were suspended in diluted AUX medium (1 : 3 in deionized water) for strain CMJ-9T, U. oligocarboniphilum DSM 21777T and U. parvum DSM 23061T or AUX medium for U. pigrum DSM 19792T. Sensitivity of strain CMJ-9T to antibiotics was tested by the disc diffusion method after spreading cell suspensions (0.5 McFarland standard) on R2A agar plates. The discs (Oxoid) contained the following antibiotics: ampicillin (10 mg), chloramphenicol (30 mg), gentamicin (10 mg), kanamycin (30 mg), nalidixic acid (30 mg), novobiocin (30 mg), penicillin G (10 U), rifampicin (5 mg), streptomycin (10 mg), sulfamethoxazole plus trimethoprim (23.75/ 1.25 mg) and tetracycline (30 mg). The effect of antibiotics on cell growth was assessed after 3 days of incubation at 25 uC. The strains were considered susceptible or resistant as described by Nokhal & Schlegel (1983). Detailed results from phenotypic and biochemical analyses of strain CMJ9T are provided in Table 1 and in the species description. Genomic DNA was isolated by using a bacterial genomic kit and the 16S rRNA gene sequence was analysed as described previously by Chen et al. (2001). The sequenced length of the 16S rRNA gene was 1422 bp for strain CMJ9T, and this gene sequence was compared to those available from EzTaxon-e (Kim et al., 2012), the Ribosomal Database Project (Cole et al., 2009) and the GenBank database http://ijs.sgmjournals.org
(http://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequence analysis was performed by using the software package BioEdit (Hall, 1999) and MEGA version 5 (Tamura et al., 2011), 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). Maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Kluge & Farris, 1969) 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. 16S rRNA gene sequence analysis indicated that strain CMJ-9T belonged to the family Oxalobacteraceae of the order Burkholderiales in the class Betaproteobacteria. Phylogenetic analyses based on the 16S rRNA gene sequence revealed that strain CMJ-9T was closely related to the species of the genera Undibacterium (94.1–96.7 % sequence similarity), Herbaspirillum (94.3–95.4 %), Oxalicibacterium (93.9–95.2 %) and Herminiimonas (94.1–94.4 %). Strain CMJ-9T formed a distinct subline within the genus Undibacterium in the neighbour-joining tree (Fig. 1). The overall topologies of the maximum-likelihood and maximumparsimony trees were similar. Sequence similarity calculations (over 1400 bp) indicated that strain CMJ-9T was closely related to U. seohonense SHS5-24T (96.7 % 16S rRNA gene sequence similarity), U. oligocarboniphilum EM 1T (95.8 %), U. jejuense JS4-4T (95.6 %), U. pigrum CCUG 49009T (94.9 %), U. parvum CCUG 49012T (94.6 %) and U. terreum C3T (94.1 %). Representative members of all other genera listed in Fig. 1 showed lower sequence similarity (,95.4 %). The DNA G+C content of strain CMJ-9T, as determined by HPLC according to Mesbah et al. (1989), was 47.7± 1.0 mol%. Isoprenoid quinones were extracted and purified according to the method of Collins (1994) and were analysed by HPLC. Strain CMJ-9T had Q-8 as the major respiratory quinone, which is the same as that of all other species of the genus Undibacterium (Ka¨mpfer et al., 2007; Eder et al., 2011; Liu et al., 2013; Kim et al., 2014). The fatty acid profiles of strain CMJ-9T, U. oligocarboniphilum DSM 21777T, U. parvum DSM 23061T and U. pigrum DSM 19792T were analysed from cells grown on R2A agar at 25 uC for 3 days. The physiological age of the different bacterial cultures at the time of harvest was standardized by choice of sector from a quadrant streak on the R2A agar plates according to the MIDI protocol (http://www. microbialid.com/PDF/TechNote_101.pdf). In this study, the different type strains of the genus Undibacterium exhibited very similar growth rates on R2A agar. Fatty acid methyl esters were prepared and separated according to the instructions of the Microbial Identification System (MIDI; Sasser, 1990) and identified by MIDI version 6.0 and the RTSBA6.00 database. Strain CMJ-9T had a fatty acid profile similar to those of all other strains of Undibacterium examined (Table 2). The predominant cellular fatty acids 1037
S.-Y. Sheu and others
Table 1. Differential characteristics of strain CMJ-9T and type strains of species of the genus Undibacterium Strains: 1, CMJ-9T; 2, U. oligocarboniphilum DSM 21777T; 3, U. parvum DSM 23061T; 4, U. pigrum DSM 19792T; 5, U. terreum C3T (data from Liu et al., 2013); 6, U. jejuense JS4-4T (Kim et al., 2014); 7, U. seohonense SHS5-24T (Kim et al., 2014). Data were obtained in this study unless indicated. +, Positive; 2, negative; W, weakly positive reaction; ND, no data available. All strains are positive for assimilation of glucose and activities of alkaline phosphatase, leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase. All strains are negative for Gram staining, indole production, assimilation of mannitol, citrate and phenylacetate and activities of C14 lipase, a-galactosidase, b-galactosidase, b-glucuronidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase. Characteristic Isolation source Colony pigmentation on R2A agar Temperature range for growth (uC) NaCl range for growth (%) Hydrolysis of: Starch Tween 80 Enzyme activities C4 esterase C8 esterase lipase Valine arylamidase Cystine arylamidase Trypsin a-Chymotrypsin a-Glucosidase Biochemical characteristics (API 20NE) Nitrate reduction Glucose acidification Arginine dihydrolase activity Urease activity Aesculin hydrolysis Gelatin hydrolysis Assimilation of (API 20NE): Arabinose Mannose N-Acetylglucosamine Maltose Gluconate Caprate Adipate Malate Acid production from (API 50CH): Ribose Galactose Fructose Cellobiose Sucrose Trehalose Gentiobiose D-Fucose DNA G+C content (mol%)
1
2
3
4
5
6
7
Freshwater pond Light yellow 10–37 0–0.8
Purified water
Drinking water
Drinking water
Soil
Soil
Freshwater
Creamy 15–30 (10–35)* 0–0.5
Yellowish 10–25 (4–30)D 0–0.5
Beige 15–35 (4–30)D 0–1.0
Creamy 15–37 0–0.8
Creamy 10–33 0–0.5
Creamy 10–30 0
2 +
+ + (2)*
2 2
+ +
2 +
2 +
2 +
+ + + + + + 2
+ + + + 2 2 +
+ + 2 2 2 2 2
+ + + 2 2 2 +
+ + + + + + 2
+ + 2 2 2 2 2
2 2 + + 2 2 2
+ 2 2 2 2 2
+ 2 2 2 2 2 (W)*
+ 2 2 2 2 2
+ 2 2 2 + +
2 + + 2 2
2 2 2 2 + 2
+ 2 2 2 2 +
2 2 + + 2 2 + +
2 2 2 + + 2 2 2
2 2 2 + 2 2 2 2
2 + + + 2 2 2 2
+ + 2 2 + + + 2
2 + 2 + 2 2 2 2
2 2 2 + 2 2 2 2
2 2 2 + + 2 2 2 47.7
+ 2 2 2 + 2 2 2 (52.4)*
+ 2 2 2 2 2 2 2 (50.6)D
+ 2 + + + + + 2 (52.3)D
2 + + + 2 2 2 + 57.4
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
W
*Results in parentheses obtained from Eder et al. (2011) DResults in parentheses obtained from Ka¨mpfer et al. (2007).
(.10 %) of strain CMJ-9T were summed feature 3 (C16 : 1v7c and/or C16 : 1v6c; 52.9 %) and C16 : 0 (28.1 %). The major cellular hydroxy fatty acid was C10 : 0 3-OH
(8.8 %). The dominant fatty acids were summed feature 3 and C16 : 0 for the novel strain and all other type strains of the genus Undibacterium, which is consistent with previous
1038
International Journal of Systematic and Evolutionary Microbiology 64
Undibacterium macrobrachii sp. nov.
Collimonas fungivorans Ter6T (AJ310394) 97 ● 100 ● 97 Collimonas pratensis Ter91T (AY281137) 98 Collimonas arenae NCCB 100031T (AY281146) Glaciimonas immobilis Cr9-30T (GU441679)
0.01 ●
74
●
Herbaspirillum autotrophicum IAM 14942T (AB074524) Oxalicibacterium solurbis MY14T (AB008503) Herminiimonas aquatilis CCUG 36956T (AM085762)
96● 98 83
Herminiimonas fonticola S-94T (AY676462) Herminiimonas arsenicoxydans ULPAs1T (CU207211)
100● Herminiimonas saxobsidens NS11T (AM493906) 97 Herminiimonas glaciei UMB49T (EU489741) ● ●
Undibacterium terreum C3T (JQ417431) Undibacterium jejuense JS4-4T (KC735150) Undibacterium parvum CCUG 49012T (AM397629)
94● 90
Undibacterium macrobrachii CMJ-9T (JQ029110) Undibacterium seohonense SHS5-24T (KC735151)
●
●
Undibacterium oligocarboniphilum EM1T (GQ379228)
●
Undibacterium pigrum CCUG 49009T (AM397630) Massilia plicata 76T (AY966000) Paucimonas lemoignei LMG 2207T (X92555) Actimicrobium antarcticum KOPRI 25157T (HQ699437)
Oxalicibacterium flavum TA17T (AY061962) 92
Oxalicibacterium faecigallinarum YOxT (AB469788) Oxalicibacterium horti OD1T (AB469786) Burkholderia acidipaludis NBRC 101816T (AB513180)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of strain CMJ-9T and related taxa in the class Betaproteobacteria. Numbers at nodes are bootstrap percentages (.70 %) based on the neighbourjoining (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 maximum-parsimony tree-making algorithms; open circles indicate that the corresponding nodes were also recovered in the tree generated with the maximum-parsimony algorithm. Burkholderia acidipaludis NBRC 101816T was used as an outgroup. Bar, 0.01 substitutions per nucleotide position.
publications (Ka¨mpfer et al., 2007; Eder et al., 2011; Liu et al., 2013; Kim et al., 2014). Furthermore, the fatty acids C12 : 0 2-OH and C17 : 0 cyclo were detected in U. terreum C3T (Liu et al., 2013) but not in strain CMJ-9T or the other type strains of Undibacterium. In contrast to strain CMJ-9T and the other type strains of the genus Undibacterium, U. terreum C3T had much higher proportions of fatty acids C10 : 0 3-OH and C12 : 0 and a much lower proportion of summed feature 3. The polar lipids of strain CMJ-9T were extracted and analysed by two-dimensional TLC according to Embley & Wait (1994). Molybdophosphoric acid was used for the detection of total polar lipids, ninhydrin for amino lipids, Zinzadze reagent for phospholipids, Dragendorff reagent for choline-containing lipids and a-naphthol reagent for glycolipids. Strain CMJ-9T exhibited a complex polar lipid profile consisting of phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylserine, two uncharacterized aminolipids and http://ijs.sgmjournals.org
three uncharacterized polar lipids (Fig. S1, available in the online Supplementary Material). The predominant polar lipids were PE, PG and DPG; the possession of PE, PG and DPG as predominant polar lipids is consistent with previous descriptions of species of Undibacterium (Ka¨mpfer et al., 2007; Eder et al., 2011; Liu et al., 2013; Kim et al., 2014). Furthermore, strain CMJ-9T differed from the other type strains of the genus Undibacterium in the presence and proportions of some minor polar lipids. Polyamines were extracted from strain CMJ-9T and analysis was carried out as described by Busse & Auling (1988) and Busse et al. (1997). Cells were homogenized in 0.2 M perchloric acid (HClO4) and centrifuged. Polyamines in the resultant supernatant were treated with dansyl chloride solution (7.5 mg ml21 in acetone) and then analysed by HPLC on a Hitachi D-7000 chromatograph and UV-Vis detector L-7420 (Hitachi). The polyamine pattern of strain CMJ-9T contained putrescine (79.9 %), 2-hydroxyputrescine 1039
S.-Y. Sheu and others
Table 2. Cellular fatty acid compositions of strain CMJ-9T and type strains of species of the genus Undibacterium Strains: 1, CMJ-9T; 2, U. oligocarboniphilum DSM 21777T; 3, U. parvum DSM 23061T; 4, U. pigrum DSM 19792T; 5, U. terreum C3T (data from Liu et al., 2013); 6, U. jejuense JS4-4T (Kim et al., 2014); 7, U. seohonense SHS5-24T (Kim et al., 2014). All data were obtained in this study with cells grown on R2A agar at 25 uC for 3 days unless indicated. Values are percentages of total fatty acids; 2, ,1.0 % or not reported. Fatty acids that make up ,1.0 % of the total in all strains are not shown. Data from Eder et al. (2011) or Ka¨mpfer et al. (2007) are given in parentheses. For unsaturated fatty acids, the position of the double bond is located by counting from the methyl (v) end of the carbon chain; cis isomers are indicated by the suffix c. Fatty acid C10 : 0 C10 : 0 3-OH C12 : 0 C12 : 0 2-OH C14 : 0 C16 : 0 C17 : 0 cyclo C18 : 0 C18 : 1v7c 11-Methyl C18 : 1v7c Summed feature 3*
1
2
3
4
5
6
7
2 8.8 5.1 2 2 28.1 2 1.7 2.9 2 52.9
1.9 (0.5) 3.5 (2.3) 3.3 (2.9) 2 (2) 1.1 (0.4) 30.7 (33.5) 2 (2) 2.3 (2) 6.7 (7.7) 2 (1.3) 49.6 (51.1)
2 (3.0) 3.6 (5.2) 3.0 (3.2) 2 (2) 2 (0.8) 30.0 (23.2) 2 (2) 2.6 (2) 10.6 (2.1) 2 (2) 49.6 (62.5)
1.1 (1.0) 3.0 (4.7) 5.2 (5.3) 2 (2) 2.1 (1.5) 25.9 (22.6) 2 (2) 1.3 (2) 10.3 (9.0) 2 (2) 50.1 (55.7)
1.7 12.9 10.2 6.9 2 25.4 1.3 2 4.3 2 36.7
2 3.2 3.2 2 2 28.3 2 2 7.9 2 50.1
2 6.3 3.2 2 2 24.5 2 2 5.4 2 58.7
*Summed features are groups of two or three fatty acids that cannot be separated by GLC using the MIDI system. Summed feature 3 comprises C16 : 1v7c and/or C16 : 1v6c.
(12.4 %), spermidine (3.5 %), homospermidine (3.0 %) and spermine (1.2 %). Putrescine and 2-hydroxyputrescine were the major polyamines of strain CMJ-9T, in line with all other species of the genus Undibacterium for which the polyamine composition has been analysed (Ka¨mpfer et al., 2007; Eder et al., 2011; Liu et al., 2013). The physiological, biochemical and morphological characteristics of strain CMJ-9T are given in the species description and Table 1. Phenotypic examination revealed many common traits between the novel strain and the other species of the genus Undibacterium. However, strain CMJ-9T could be clearly differentiated from U. oligocarboniphilum DSM 21777T, U. parvum DSM 23061T, U. pigrum DSM 19792T, U. terreum C3T, U. jejuense JS4-4T and U. seohonense SHS5-24T by its colony pigmentation and by some enzyme activities and biochemical characteristics (Table 1).
Description of Undibacterium macrobrachii sp. nov. Undibacterium macrobrachii (ma.cro.bra9chi.i. N.L. n. Macrobrachium the scientific name of a genus of shrimp; N.L. gen. n. macrobrachii of Macrobrachium, referring to the isolation of the type strain from freshwater around shrimp belonging to the genus Macrobrachium).
Cells of strain CMJ-9T were Gram-staining-negative, oxidase-positive, motile by a single polar flagellum and chemoheterotrophic, growing under mesophilic and neutrophilic conditions. The quinone system, polyamine pattern, major characteristics of the polar lipid and fatty acid profiles and 16S rRNA gene sequence analysis suggested the placement of strain CMJ-9T in the genus Undibacterium. The phylogenetic inference is supported by the unique combination of chemotaxonomic and biochemical characteristics of the novel strain. Hence, strain CMJ-9T constitutes a member of the genus Undibacterium. The name Undibacterium macrobrachii sp. nov. is proposed for this taxon.
Cells are strictly aerobic, Gram-negative, flagellated and rod-shaped. Poly-b-hydroxybutyrate accumulation is observed. After 72 h of incubation on R2A agar at 25 uC, the mean cell size is 0.3–0.561.4–2.0 mm. Colonies on R2A agar are light yellow, circular, smooth and convex with entire edges. Colonies are approximately 1.0–1.3 mm in diameter on R2A agar after 72 h of incubation at 25 uC. Growth occurs at 10–37 uC, with 0–0.8 % NaCl and at pH 6.0–9.0. Optimum growth occurs at 20–30 uC, with 0–0.1 % NaCl and at pH 6.0–7.0. Positive for oxidase activity and hydrolysis of Tweens 20 and 80. Negative for catalase, DNase and lipase (corn oil) activities and hydrolysis of casein, starch, CM-cellulose and Tweens 40 and 60. In API 20NE tests, positive for nitrate reduction and assimilation of glucose, N-acetylglucosamine, maltose, adipate and malate and negative for indole production, fermentation of D-glucose, activities of arginine dihydrolase, urease and b-galactosidase, hydrolysis of aesculin and gelatin and assimilation of arabinose, mannose, mannitol, gluconate, caprate, citrate and phenylacetate. In API ZYM tests, alkaline phosphatase, C4 esterase, C8 esterase lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, acid phosphatase and
1040
International Journal of Systematic and Evolutionary Microbiology 64
Undibacterium macrobrachii sp. nov.
naphthol-AS-BI-phosphohydrolase activities are present; C14 lipase, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase activities are absent. The following compounds are utilized in the API 50CH test system: glucose, N-acetylglucosamine, cellobiose, maltose, sucrose, starch and glycogen. Negative for assimilation of glycerol, erythritol, D- and L-arabinose, ribose, D- and L-xylose, adonitol, methyl b-xyloside, galactose, fructose, mannose, sorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, methyl a-D-mannoside, methyl a-glucoside, amygdalin, arbutin, aesculin, salicin, lactose, melibiose, trehalose, inulin, melezitose, raffinose, xylitol, gentiobiose, turanose, lyxose, tagatose, D- and L-fucose, D- and L-arabitol, gluconate, 2-ketogluconate and 5ketogluconate. Sensitive to tetracycline, sulfamethoxazole plus trimethoprim, rifampicin, novobiocin, streptomycin, chloramphenicol, nalidixic acid, penicillin G, ampicillin, gentamicin and kanamycin. The predominant cellular fatty acids are summed feature 3 (C16 : 1v7c and/or C16 : 1v6c) and C16 : 0. The major cellular hydroxy fatty acid is C10 : 0 3-OH. The polar lipid profile consists of the predominant lipids phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The polyamine profile is composed of the major compound putrescine and moderate amounts of 2-hydroxyputrescine. The major respiratory quinone is Q-8. The type strain is CMJ-9T (5BCRC 80406T5LMG 26891T 5KCTC 23916T), isolated from a freshwater shrimp culture pond in the Pingtung countryside of southern Taiwan. The DNA G+C content of the type strain is 47.7 mol%.
Cole, J. R., Wang, Q., Cardenas, E., Fish, J., Chai, B., Farris, R. J., Kulam-Syed-Mohideen, A. S., McGarrell, D. M., Marsh, T. & other authors (2009). The Ribosomal Database Project: improved
alignments and new tools for rRNA analysis. Nucleic Acids Res 37, D141–D145. Collins, M. D. (1994). Isoprenoid quinones. In Chemical Methods in
Prokaryotic Systematics, pp. 265–309. Edited by M. Goodfellow & A. G. O’Donnell. Chichester: Wiley. Eder, W., Wanner, G., Ludwig, W., Busse, H.-J., Ziemke-Ka¨geler, F. & Lang, E. (2011). Description of Undibacterium oligocarboniphilum
sp. nov., isolated from purified water, and Undibacterium pigrum strain CCUG 49012 as the type strain of Undibacterium parvum sp. nov., and emended descriptions of the genus Undibacterium and the species Undibacterium pigrum. Int J Syst Evol Microbiol 61, 384–391. Embley, T. M. & Wait, R. (1994). Structural lipids of eubacteria. In Chemical Methods in Prokaryotic Systematics, pp. 121–161. Edited by M. Goodfellow & A. G. O’Donnell. Chichester: Wiley. Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a
maximum likelihood approach. J Mol Evol 17, 368–376. PHYLIP (phylogeny inference package), version 3.5c. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle, USA.
Felsenstein, J. (1993).
Garrity, G. M., Bell, J. A. & Lilburn, T. (2005). Family II. Oxalobac-
teraceae fam. nov. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2C, p. 623. Edited by D. J. Brenner, N. R. Krieg, J. T. Staley & G. M. Garrity. New York: Springer. Hall, T. A. (1999). BioEdit: a user-friendly biological sequence align-
ment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41, 95–98. Ka¨mpfer, P., Rossello´-Mora, R., Hermansson, M., Persson, F., Huber, B., Falsen, E. & Busse, H.-J. (2007). Undibacterium pigrum
gen. nov., sp. nov., isolated from drinking water. Int J Syst Evol Microbiol 57, 1510–1515. 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
References Beveridge, T. J., Lawrence, J. R. & Murray, R. G. E. (2007). Sampling
EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62, 716–721.
and staining for light microscopy. In Methods for General and Molecular Bacteriology, 3rd edn, pp. 19–33. Edited by T. J. Beveridge, J. A. Breznak, G. A. Marzluf, T. M. Schmidt & L. R. Snyder. Washington, DC: American Society for Microbiology.
Kim, S. J., Moon, J. Y., Weon, H. Y., Hong, S. B., Seok, S. J. & Kwon, S. W. (2014). Undibacterium jejuense sp. nov. and Undibacterium
Bowman, J. P. (2000). Description of Cellulophaga algicola sp. nov.,
Kimura, M. (1983). The Neutral Theory of Molecular Evolution. Cambridge: Cambridge University Press.
isolated from the surfaces of Antarctic algae, and reclassification of Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Cellulophaga uliginosa comb. nov. Int J Syst Evol Microbiol 50, 1861–1868. Breznak, J. A. & Costilow, R. N. (2007). Physicochemical factors in
growth. In Methods for General and Molecular Bacteriology, 3rd edn, pp. 309–329. Edited by T. J. Beveridge, J. A. Breznak, G. A. Marzluf, T. M. Schmidt & L. R. Snyder. Washington, DC: American Society for Microbiology. Busse, H.-J. & Auling, G. (1988). Polyamine pattern as chemotaxo-
nomic marker within the Proteobacteria. Syst Appl Microbiol 11, 1–8.
seohonense sp. nov., isolated from soil and freshwater, respectively. Int J Syst Evol Microbiol (in press).
Kluge, A. G. & Farris, F. S. (1969). Quantitative phyletics and the
evolution of anurans. Syst Zool 18, 1–32. Liu, Y. Q., Wang, B. J., Zhou, N. & Liu, S. J. (2013). Undibacterium
terreum sp. nov., isolated from permafrost soil. Int J Syst Evol Microbiol 63, 2296–2300. Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise
measurement of the G+C content of deoxyribonucleic acid by highperformance liquid chromatography. Int J Syst Bacteriol 39, 159–167. Nokhal, T. H. & Schlegel, H. G. (1983). Taxonomic study of
Busse, H.-J., Bunka, S., Hensel, A. & Lubitz, W. (1997). Discrimina-
Paracoccus denitrificans. Int J Syst Bacteriol 33, 26–37.
tion of members of the family Pasteurellaceae based on polyamine patterns. Int J Syst Bacteriol 47, 698–708.
Powers, E. M. (1995). Efficacy of the Ryu nonstaining KOH technique
for rapidly determining gram reactions of food-borne and waterborne bacteria and yeasts. Appl Environ Microbiol 61, 3756–3758.
Chen, W. M., Laevens, S., Lee, T. M., Coenye, T., De Vos, P., Mergeay, M. & Vandamme, P. (2001). Ralstonia taiwanensis sp. nov., isolated
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a
from root nodules of Mimosa species and sputum of a cystic fibrosis patient. Int J Syst Evol Microbiol 51, 1729–1735.
new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.
http://ijs.sgmjournals.org
1041
S.-Y. Sheu and others Sasser, M. (1990). Identification of bacteria by gas chromatography of
cellular fatty acids, Technical Note no. 101. Newark, DE: MIDI Inc.
maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739.
Schlegel, H. G., Lafferty, R. & Krauss, I. (1970). The isolation of mutants not accumulating poly-b-hydroxybutyric acid. Arch Mikrobiol
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible
71, 283–294.
strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Spiekermann, P., Rehm, B. H. A., Kalscheuer, R., Baumeister, D. & Steinbu¨chel, A. (1999). A sensitive, viable-colony staining method
using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Arch Microbiol 171, 73–80.
Tindall, B. J., Sikorski, J., Smibert, R. A. & Krieg, N. R. (2007).
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using
Phenotypic characterization and the principles of comparative systematics. In Methods for General and Molecular Bacteriology, 3rd edn, pp. 330–393. Edited by T. J. Beveridge, J. A. Breznak, G. A. Marzluf, T. M. Schmidt & L. R. Snyder. Washington, DC: American Society for Microbiology.
1042
International Journal of Systematic and Evolutionary Microbiology 64
