Antonie van Leeuwenhoek (2015) 108:695–701 DOI 10.1007/s10482-015-0525-0
ORIGINAL PAPER
Hydrogenophaga luteola sp. nov. isolated from reed pond water Juan Du . Jung-Eun Yang . Hina Singh . Shahina Akter . KyungHwa Won . Chang Shik Yin . Feng-Xie Jin . Tae-Hoo Yi
Received: 14 April 2015 / Accepted: 30 June 2015 / Published online: 8 July 2015 Ó Springer International Publishing Switzerland 2015
Abstract A yellowish colored, Gram-staining negative, strictly aerobic, motile, rod-shaped bacterium, designated THG-SQE7T, was isolated from reed pond water in Shangqiu, PR China. Comparative 16S rRNA gene sequence analysis indicated that strain THGSQE7T is most closely related to Hydrogenophaga pseudoflava ATCC 33668T (98.4 %), followed by Hydrogenophaga bisanensis K102T (97.6 %) and Hydrogenophaga flava CCUG 1658T (97.6 %). DNA–DNA hybridization showed 53.5, 36.0 and The NCBI GenBank accession number for the 16S rRNA gene sequence of strains THG-SQE7T is KM598239.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0525-0) contains supplementary material, which is available to authorized users. J. Du J.-E. Yang H. Singh S. Akter K. Won T.-H. Yi (&) Department of Oriental Medicinal Biotechnology, College of Life Science, Kyung Hee University, Global Campus, 1732 Deokyoungdaero, Giheung-gu, Yongin-si, Gyeonggi-do 446-701, Republic of Korea e-mail: [email protected] C. S. Yin Department of Acupuncture Merdian Science Research Center, College of Korean Medicine, Kyung Hee University, Global Campus, Seoul, Republic of Korea F.-X. Jin College of Bio and Food Technology, Dalian Polytechnic University, Qinggong-yuan No. 1, Ganjingzi-qu, Dalian 116034, People’s Republic of China
22.5 % DNA re-association with H. pseudoflava KCTC 2348T, H. bisanensis KCTC 12980T and H. flava KCTC 1648T, respectively. Chemotaxonomic data revealed that strain THG-SQE7T possesses ubiquinone-8 as the only isoprenoid quinone, summed feature 3 (C16:1x7c and/or C16:1x6c), C16:0 and C18:1x7c as the major fatty acids. The major polar lipids were found to be phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The DNA G?C content was determined to be 63.7 mol%. These data corroborated the affiliation of strain THGSQE7T to the genus Hydrogenophaga. Thus, the isolate represents a novel species, for which the name Hydrogenophaga luteola sp. nov. is proposed, with THG-SQE7T as the type strain (=KCTC 42501T = CCTCC AB 2014314T = JCM 30433T). Keywords Hydrogenophaga luteola Gramstaining negative Ubiquinone-8 16S rRNA
Introduction The genus Hydrogenophaga, which belongs to the family Comamonadaceae, was first described by Willems et al. (1989) to accommodate several misclassified Pseudomonas species including Hydrogenophaga flava, isolated from ditch mud; H. pseudoflava isolated from soil, mud, or water; H. taeniospiralis isolated from soil and H. palleronii isolated from water. Subsequently, five more species
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H. intermedia (Contzen et al. 2000); H. defluvii and H. atypica (Ka¨mpfer et al. 2005); H. caeni (Chung et al. 2007) and H. bisanensis (Yoon et al. 2008) isolated from wastewater and activated sludge have been also described. The names of another three species isolated from compost, anodic biofilms and mixed culture, H. temperata (Kim et al. 2010), H. electricum (Kimura and Okabe 2013) and H. carboriunda (Reinauer et al. 2014), respectively, are not yet validated. Strains of the genus Hydrogenophaga are Gram-staining negative, oxidase positive, catalase variable and rodshaped bacteria with high G?C content (61.6–70.4 mol%). Bacteria of the genus Hydrogenophaga contain ubiquinone 8 (Q-8), are motile by means of one, rarely two, polar or subpolar flagella, chemo-organotrophic or chemolithoautotrophic, using the oxidation of H2 as an energy source and CO2 as a carbon source (Willems et al. 1989; Ka¨mpfer et al. 2005; Chung et al. 2007; Yoon et al. 2008; Kimura and Okabe 2013). Here, we report on the taxonomic characterization of a Hydrogenophaga-like bacterial strain, THG-SQE7T, which was isolated from a reed pond water sample in Shangqiu, PR Chian.
Materials and methods Isolation of bacterial strain Sample was collected from reed (Phragmites australis) pond water in Shangqiu (34°220 3900 N, 115°390 3500 E), Henan Province, PR China. Strain THG-SQE7T was isolated by conventional dilution–plating method using Reasoner’s 2A agar (R2A agar; BD, U.S.A). The isolate was routinely grown on R2A agar at 28 °C and stored in nutrient broth containing glycerol suspension (25 %, w/v) at -80 °C. Reference type strains H. pseudoflava KCTC 2348T, H. bisanensis KCTC 12980T and H. flava KCTC 1648T were obtained from Korean Collection for Type Cultures (KCTC) and used in pheno- and chemotypic analysis for comparative purposes. Morphological and physiological characterization Cells morphology was observed after 2 days growth at 28 °C on R2A agar. The cells were placed on carbonand Formvar-coated nickel grids and stained with 0.1 % (w/v) aqueous uranyl acetate and observed by using
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transmission electron microscope (Model JEM1010; JEOL) at 911,000 magnification under standard operating conditions. Gram-staining was determined using a bioMe´rieux (France) Gram stain kit. Growth on nutrient agar (NA, Oxoid), tryptone soya agar (TSA, Oxoid), Luria–Bertani agar (LBA; Oxoid) and MacConkey Agar (Oxoid) was also tested. Motility test was performed by using sulfide–indole–motility medium (SIM; BD). Anaerobic growth was tested in R2A broth supplemented with thioglycollate [0.1 % (w/v), Sigma]. Growth at different temperatures (4, 10, 15, 18, 25, 28, 30, 35, 37 and 45 °C) and pH conditions (pH 4.0–10.0, at intervals of 0.5 pH units) were determined in R2A broth. Two different buffers were used (final concentration, 100 mM): acetate buffer was used for pH 4.0–6.5 and phosphate buffer was used for pH 7.0–10.0. Salt tolerance was tested in R2A broth containing 0–5 % (w/v) NaCl (at intervals of 0.5 %). Growth was estimated by monitoring the optical density at 600 nm. Oxidase and catalase activity were tested with 1 % (w/v) N, N, N0 , N0 -tetramethyl-1, 4-phenylenediamine reagent and 3 % (v/v) H2O2, respectively. Tests for hydrolysis of DNA (DNase agar, Oxoid), casein [on R2A agar supplemented with 2 % skim milk (Oxoid)], starch [on R2A agar containing 1 % starch (Difco)], carboxymethyl-cellulose (CMC) [on R2A agar containing 0.1 % CMC (Sigma)], L-tyrosine [on R2A agar containing 0.5 % L-tyrosine (Sigma)], chitin [on R2A agar containing 1 % chitin from crab shell (Sigma)], esculin [on R2A agar containing 0.1 % esculin and 0.02 % ferric citrate (Difco)], Tween 20 [on R2A agar containing 0.01 % CaCl22H2O and 1 % Tween 20 (Sigma)] and Tween 80 [on R2A agar containing 0.01 % CaCl22H2O and 1 % Tween 80 (Sigma)] were performed. Chemolithoautotrophic growth of strain THGSQE7T with hydrogen gas was tested on Medium 81 agar (http://www.dsmz.de/microorganisms/medium/ pdf/DSMZ_Medium81.pdf) under the conditions described by Malik and Schlegel (1981). In the above description, except specially indicated, all the tests were evaluated after 7 days of incubation at 28 °C. In addition, API 20NE and API ZYM tests (bioMe´rieux) were used for evaluating basic chemical test, carbon source assimilation and enzyme activity, according to the manufacturer’s instructions. Strains H. pseudoflava KCTC 2348T, H. bisanensis KCTC 12980T and H. flava KCTC 1648T were included as references for the investigation of the biochemical tests using the same laboratory conditions.
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Molecular characterization and phylogenetic construction
values were converted to percentage DNA–DNA relatedness values.
Genomic DNA was extracted and purified using a Genomic DNA extraction kit (Solgent, Korea). The 16S rRNA gene was amplified from the chromosomal DNA with the universal bacterial primer pair 27F and 1492R according to the methods of Weisburg et al. (1991). Sequencing of 16S rRNA gene was performed by Solgent Co. Ltd (Korea). Sequences of related taxa were obtained from GenBank database and EzTaxon-e server (http://www.ezbiocloud.net/eztaxon; Kim et al. 2012). Multiple alignments were performed using CLUSTAL_X (Thompson et al. 1997), gap editions were performed using BioEdit program (Hall 1999). The neighbor-joining (Saitou and Nei 1987), maximum-likelihood (Felsenstein 1981) and maximumparsimony (Fitch 1971) methods were used to construct phylogenetic trees in MEGA 6 program package (Kumar et al. 2008; Tamura et al. 2013). All the species of the genus Hydrogenophaga were included in the phylogenetic trees. The Kimura two-parameter model (Kimura 1983) was used to calculate the evolutionary distances. The bootstrap values were calculated based on 1000 replications (Felsenstein 1985).
Chemotaxonomic characterization
DNA G?C content and DNA–DNA hybridization Genomic DNA of strain THG-SQE7T and the three closest reference strains were extracted as described elsewhere (Moore and Dowhan 1995). Genomic DNA of strain THG-SQE7T and Escherichia coli strain B (Sigma-Aldrich D4889) was degraded enzymatically into nucleosides by nuclease P1 and alkaline phosphatase (Sigma). The DNA G?C content was analyzed using reverse-phase HPLC system (Alliance 2690 system, Waters) as described by Mesbah et al. (1989), the E. coli strain B was used as standard. DNA–DNA hybridization was performed between strain THG-SQE7T and H. pseudoflava KCTC 2348T, H. bisanensis KCTC 12980T and H. flava KCTC 1648T, using photobiotin-labelled probes in microplate wells (Ezaki et al. 1989). Optimum hybridization temperature was at 43 °C. Hybridization was performed with five replications for each sample. The highest and lowest values obtained for each sample were excluded and the means of the remaining three
For quinone and polar lipids analysis, cells were grown in R2A broth at 28 °C for 3 days, collected by centrifugation and freeze-dried. Isoprenoid quinones analysis of strain THG-SQE7T and its closest reference strain H. pseudoflava KCTC 2348T was performed using RP-HPLC Waters 2690 Alliance system as previously described (Collins and Jones 1981; Hiraishi et al. 1996). Polar lipids of strains THGSQE7T and H. pseudoflava KCTC 2348T were analyzed by using two-dimensional thin layer chromatography (Minnikin et al. 1984). The lipids were visualized using the following reagents: 5 % molybdatophosphoric acid (total lipids, Sigma), 0.2 % ninhydrin (aminolipids, Sigma), and 2.5 % anaphthol-sulfuric acid (glycolipids, Sigma) and molybdenum blue (phospholipids, Sigma). For fatty acid analysis, strain THG-SQE7T and the three closest reference strains were grown on R2A agar at 28 °C for 2 days (the type strains exhibited similar growth rates). The cellular fatty acid profiles were analysed according to the protocol of Sherlock Microbial Identification System (MIDI) and identified with GC (Hewlett Packard 6890) using Sherlock Aerobic Bacterial Database (TSBA60) (Sasser 1990).
Results and discussion Phenotypic analysis showed that cells of strain THGSQE7T were Gram-staining negative, strictly aerobic and rod-shaped, approximately 0.60–0.70 9 1.85–2.40 lm, and motile by means of a polar flagellum (Supplementary Fig. S2), which are typical characteristics for bacteria of the genus Hydrogenophaga (Willems et al. 1989). The comparative analysis of biochemical and physiological characteristics of strain THG-SQE7T and H. pseudoflava KCTC 2348T, H. bisanensis KCTC 12980T and H. flava KCTC 1648T indicated that all strains are motile, positive for nitrate reduction, negative for hydrolysis of starch, gelatin and chitin. All strains are positive for esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase
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activity; negative for cystine arylamidase, trypsin, achymotrypsin, a-galactosidase, b-glucuronidase, bgalactosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase activity. All strains are negative for indole production, glucose acidification, and assimilation of adipic acid, capric acid, citric acid and phenylacetic acid; positive for assimilation of D-glucose and gluconate. The distinction between strain THG-SQE7T and the three closely related reference strains were in production of lipase, urease, b-glucosidase, casein hydrolysis and Nacetyl-glucosamine; THG-SQE7T is unable to hydrolyse the DNA, in contrast to the other three strains (other details are given in Table 1). Phenotypic and biochemical properties suggested that stain THG-SQE7T is clearly distinguished form previously described species of the genus Hydrogenophaga and may represent a novel species. The phylogenetic analysis based on the 16S rRNA gene sequence of strain THG-SQE7T (NCBI accession number is KM598239) confirmed that strain THGSQE7T belongs to the genus Hydrogenophaga, and is most closely related to H. pseudoflava ATCC 33668T (98.4 %), followed by H. bisanensis K102T (97.6 %) and H. flava CCUG 1658T (97.6 %), while 16S rRNA gene sequence similarity with other species of the genus were found to be below 96.6 % (Fig. 1, Supplementary Fig. S1). According to genetic analysis, strain THGSQE7T showed 53.5, 36.0 and 22.5 % DNA–DNA relatedness with H. pseudoflava KCTC 2348T, H. bisanensis KCTC 12980T and H. flava KCTC 1648T, respectively. The DNA relatedness values between strain THG-SQE7T and three most phylogenetically closely related strains were found to be significantly lower than the recommended threshold of 70 % (Wayne et al. 1987; Stackebrandt and Goebel 1994). The DNA G?C content of the novel isolate was 63.7 mol%, which conforms to the expected range of G?C contents for the genus Hydrogenophaga. The polar lipid profiles of strain THG-SQE7T and H. pseudoflava KCTC 2348T are shown in Supplementary Fig. S3. The polar lipid profile of strain THGSQE7T was found to be different to that of H. pseudoflava KCTC 2348T by the presence of unidentified lipids (L4 and L5) and by the lack of unidentified phospholipid (PL1) and an unidentified lipid (L0). Strain THG-SQE7T contains Q-8 as the only respiratory quinone, which is a typical characteristic for bacteria of the genus Hydrogenophaga (Willems et al.
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Antonie van Leeuwenhoek (2015) 108:695–701 Table 1 Physiological characteristics of THG-SQE7T and the related species of the genus Hydrogenophaga Characteristics
1
2
3
4
Hydrolysis of: Esculin
?
?
-
-
L-Tyrosine
-
-
?
-
Casein
?
-
-
-
CMC
?
?
-
?
DNA
-
?
?
?
Tween 20
-
?
-
-
Tween 80
-
?
-
-
D-maltose
?
?
-
?
D-mannitol
?
?
-
-
Assimilation of
D-Mannose
?
?
-
-
L-Arabinose
?
?
-
-
Malic acid
?
?
-
?
?
-
-
-
Urease
?
-
-
-
Alkaline phosphatase
-
?
-
?
Lipase (C14)
W
-
-
-
Valine arylamidase
?
?
-
-
a-glucosidase
?
?
-
?
b-glucosidase
?
-
-
-
?
-
?
N-Acetyl-glucosamine Enzyme activities
Arginine dihydrolase T
T
Strains 1 THG-SQE7 , 2 H. pseudoflava KCTC 2348 , 3 H. bisanensis KCTC 12980T, 4 H. flava KCTC 2348T (type strain of the genus) All data was carried out in this study ? positive, - negative, w weakly positive
1989). The major fatty acids of strain THG-SQE7T were identified as summed feature 3 (C16:1x7c and/or C16:1x6c) (51.9 %), C16:0 (22.5 %) and C18:1x7c (12.2 %). The fatty acid composition of strain THG-SQE7T was very similar to H. pseudoflava KCTC 2348T, H. bisanensis KCTC 12980T and H. flava KCTC 2348T (Table 2). The phylogenetic analysis based on the 16S rRNA gene sequence similarity, phenotypic and chemotaxonomic properties revealed that strain THG-SQE7T can be clearly differentiated from other members of the genus Hydrogenophaga and represents a novel species, for which the name Hydrogenophaga luteola sp. nov. is proposed.
Antonie van Leeuwenhoek (2015) 108:695–701
98
699
Hydrogenophaga taeniospiralis ATCC 49743T (AF078768)
91
*Hydrogenophaga
carboriunda YZ2T (EU095331)
Hydrogenophaga palleronii DSM 63T (AF019073)
91
T 100 Hydrogenophaga atypica BSB 41.8 (AJ585992)
83
Hydrogenophaga defluvii BSB 9.5T (AJ585993) Hydrogenophaga ntermedia S1T (AF019037) Hydrogenophaga bisanensis K102T (EF532793)
74
Hydrogenophaga flava CCUG 1658T (AF078771) Hydrogenophaga luteola THG-SQE7T (KM598239)
83
92
86
Hydrogenophaga pseudoflava ATCC 33668T (AF078770) * Hydrogenophaga * Hydrogenophaga
83
97
temperata TR7-01T (AB166886)
electricum AR20T (AB746948)
Malikia granosa P1T (AJ627188) Malikia spinosa ATCC 14606T (AB021387) Macromonas bipunctata IAM 14880T (AB077037) Tepidicella xavieri TU-16T (DQ295805) Hydrogenophaga caeni EMB71T (DQ372983) Xenophilus aerolatus 5516S-2T (EF660342) Brachymonas chironomi AIMA4T (EU346912) Diaphorobacter oryzae RF3T (EU342381)
Delftia litopenaei wsw-7T (GU721027) Comamonas odontotermitis Dant 3-8T (DQ453128) Comamonas badia DSM 17552T (AXVM01000023) 0.005 Fig. 1 Neighbor-joining tree based on 16S rRNA gene sequence analysis, showing relationships between strain THGSQE7T and related members of the genus Hydrogenophaga. Bootstrap values (expressed as percentage of 1000 replications)
over 70 % are shown at the branching points. Comamonas badia DSM 17552T (AXVM01000023) was used as outgroup. Bar 0.005 substitutions per nucleotide position. Symbol * indicated the non-valid species of genus Hydrogenophaga
Description of Hydrogenophaga luteola sp. nov
not on LBA or MacConkey agar. Catalase and oxidase positive, able to hydrolyze esculin, CMC and casein, but not able to hydrolyze chitin, gelatin, starch, Tween 20, Tween 80 or L-tyrosine. Shows chemolithotrophic growth, using the oxidation of H2 as an energy source. Positive for esterase lipase (C8), esterase (C4), leucine arylamidase, valine arylamidase, acid phosphatase, aglucosidase, b-glucosidase and naphthol-AS-BI-phosphohydrolase; negative for alkaline phosphatase, agalactosidase, cystine arylamidase, trypsin, a-chymotrypsin, a-fucosidase, a-mannosidase, b-galactosidase, b-glucuronidase and N-acetyl-bglucosaminidase; weakly positive for lipase (C14).
Hydrogenophaga luteola (lu.te’o.la. L. masc. adj. luteola yellowish, referring to the colour of the colonies on R2A agar) Cells are Gram-staining negative, strictly aerobic, motile by means of a polar flagellum and rod-shaped (0.60–0.70 9 1.85–2.40 lm). Colonies are round, yellowish, non-sticky, translucent and convex with entire margins on R2A agar. Growth occurs at 18–37 °C (optimum, 28–37 °C); at pH 6.0–9.0 (optimum, 6.5–8.0) and at 0–2.0 % NaCl (optimum, 0–0.5 %). Grows on R2A agar, NA and TSA, but do
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Antonie van Leeuwenhoek (2015) 108:695–701
Table 2 Cellular fatty acid profiles of THG-SQE7T and the related species of the genus Hydrogenophaga Fatty acid
1
2
3
4
4.2
Straight C14:0
4.3
3.5
Tr
C16:0
22.5
25.4
23.3
23.1
C17:0
Tr
1.8
1.0
1.6
C18:0
Tr
Tr
2.7
Tr
Unsaturated C17:1 x6c
Tr
1.2
2.0
1.6
C18:1 x7c
12.2
15.6
17.7
18.9
C19:1 x6c
ND
ND
3.3
ND
Tr
ND
1.0
ND
C10:0 3OH
4.5
3.5
ND
3.6
C18:0 3OH
ND
ND
Tr
ND
51.9
45.8
42.4
43.4
Branched-chain Anteiso-C12:0 Hydroxy
Summed feature 3a
Strains 1 THG-SQE7T, 2 H. pseudoflava KCTC 2348T, 3 H. bisanensis KCTC 12980T, 4 H. flava KCTC 2348T All data was carried out in this study Fatty acids of less than 0.5 % in all strains were not listed ND not detected, Tr traces (\1.0 %) Summed feature 3a consisted of C16:1x7c/C16:1x6c
The results of indole production, glucose acidification, protease activity and assimilation of adipic acid, capric acid, phenylacetic acid and citric acid are negative; the results of nitrate reduction, b-glucosidase, arginine dihydrolase, urease activity, D-maltose, D-mannitol, D-glucose, L-arabinose, D-mannose, Nacetyl-glucosamine, gluconate and malic acid assimilation are positive. The polar lipid profiles consist of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and five unidentified lipids. The major fatty acids are summed feature 3 (C16:1x7c and/or C16:1x6c), C16:0 and C18:1x7c. Contains Q-8 as the only respiratory quinone. The DNA G?C content of the type strain is 63.7 mol%. The type strain, THG-SQE7T (=KCTC 42501T = CCTCC AB 2014314T = JCM 30433T), was isolated from reed pond water in Shangqiu, PR China. Acknowledgments This work was conducted under the industrial infrastructure program (No. N0000888) for fundamental technologies which is funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea).
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ORIGINAL PAPER
Hydrogenophaga luteola sp. nov. isolated from reed pond water Juan Du . Jung-Eun Yang . Hina Singh . Shahina Akter . KyungHwa Won . Chang Shik Yin . Feng-Xie Jin . Tae-Hoo Yi
Received: 14 April 2015 / Accepted: 30 June 2015 / Published online: 8 July 2015 Ó Springer International Publishing Switzerland 2015
Abstract A yellowish colored, Gram-staining negative, strictly aerobic, motile, rod-shaped bacterium, designated THG-SQE7T, was isolated from reed pond water in Shangqiu, PR China. Comparative 16S rRNA gene sequence analysis indicated that strain THGSQE7T is most closely related to Hydrogenophaga pseudoflava ATCC 33668T (98.4 %), followed by Hydrogenophaga bisanensis K102T (97.6 %) and Hydrogenophaga flava CCUG 1658T (97.6 %). DNA–DNA hybridization showed 53.5, 36.0 and The NCBI GenBank accession number for the 16S rRNA gene sequence of strains THG-SQE7T is KM598239.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0525-0) contains supplementary material, which is available to authorized users. J. Du J.-E. Yang H. Singh S. Akter K. Won T.-H. Yi (&) Department of Oriental Medicinal Biotechnology, College of Life Science, Kyung Hee University, Global Campus, 1732 Deokyoungdaero, Giheung-gu, Yongin-si, Gyeonggi-do 446-701, Republic of Korea e-mail: [email protected] C. S. Yin Department of Acupuncture Merdian Science Research Center, College of Korean Medicine, Kyung Hee University, Global Campus, Seoul, Republic of Korea F.-X. Jin College of Bio and Food Technology, Dalian Polytechnic University, Qinggong-yuan No. 1, Ganjingzi-qu, Dalian 116034, People’s Republic of China
22.5 % DNA re-association with H. pseudoflava KCTC 2348T, H. bisanensis KCTC 12980T and H. flava KCTC 1648T, respectively. Chemotaxonomic data revealed that strain THG-SQE7T possesses ubiquinone-8 as the only isoprenoid quinone, summed feature 3 (C16:1x7c and/or C16:1x6c), C16:0 and C18:1x7c as the major fatty acids. The major polar lipids were found to be phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The DNA G?C content was determined to be 63.7 mol%. These data corroborated the affiliation of strain THGSQE7T to the genus Hydrogenophaga. Thus, the isolate represents a novel species, for which the name Hydrogenophaga luteola sp. nov. is proposed, with THG-SQE7T as the type strain (=KCTC 42501T = CCTCC AB 2014314T = JCM 30433T). Keywords Hydrogenophaga luteola Gramstaining negative Ubiquinone-8 16S rRNA
Introduction The genus Hydrogenophaga, which belongs to the family Comamonadaceae, was first described by Willems et al. (1989) to accommodate several misclassified Pseudomonas species including Hydrogenophaga flava, isolated from ditch mud; H. pseudoflava isolated from soil, mud, or water; H. taeniospiralis isolated from soil and H. palleronii isolated from water. Subsequently, five more species
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H. intermedia (Contzen et al. 2000); H. defluvii and H. atypica (Ka¨mpfer et al. 2005); H. caeni (Chung et al. 2007) and H. bisanensis (Yoon et al. 2008) isolated from wastewater and activated sludge have been also described. The names of another three species isolated from compost, anodic biofilms and mixed culture, H. temperata (Kim et al. 2010), H. electricum (Kimura and Okabe 2013) and H. carboriunda (Reinauer et al. 2014), respectively, are not yet validated. Strains of the genus Hydrogenophaga are Gram-staining negative, oxidase positive, catalase variable and rodshaped bacteria with high G?C content (61.6–70.4 mol%). Bacteria of the genus Hydrogenophaga contain ubiquinone 8 (Q-8), are motile by means of one, rarely two, polar or subpolar flagella, chemo-organotrophic or chemolithoautotrophic, using the oxidation of H2 as an energy source and CO2 as a carbon source (Willems et al. 1989; Ka¨mpfer et al. 2005; Chung et al. 2007; Yoon et al. 2008; Kimura and Okabe 2013). Here, we report on the taxonomic characterization of a Hydrogenophaga-like bacterial strain, THG-SQE7T, which was isolated from a reed pond water sample in Shangqiu, PR Chian.
Materials and methods Isolation of bacterial strain Sample was collected from reed (Phragmites australis) pond water in Shangqiu (34°220 3900 N, 115°390 3500 E), Henan Province, PR China. Strain THG-SQE7T was isolated by conventional dilution–plating method using Reasoner’s 2A agar (R2A agar; BD, U.S.A). The isolate was routinely grown on R2A agar at 28 °C and stored in nutrient broth containing glycerol suspension (25 %, w/v) at -80 °C. Reference type strains H. pseudoflava KCTC 2348T, H. bisanensis KCTC 12980T and H. flava KCTC 1648T were obtained from Korean Collection for Type Cultures (KCTC) and used in pheno- and chemotypic analysis for comparative purposes. Morphological and physiological characterization Cells morphology was observed after 2 days growth at 28 °C on R2A agar. The cells were placed on carbonand Formvar-coated nickel grids and stained with 0.1 % (w/v) aqueous uranyl acetate and observed by using
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transmission electron microscope (Model JEM1010; JEOL) at 911,000 magnification under standard operating conditions. Gram-staining was determined using a bioMe´rieux (France) Gram stain kit. Growth on nutrient agar (NA, Oxoid), tryptone soya agar (TSA, Oxoid), Luria–Bertani agar (LBA; Oxoid) and MacConkey Agar (Oxoid) was also tested. Motility test was performed by using sulfide–indole–motility medium (SIM; BD). Anaerobic growth was tested in R2A broth supplemented with thioglycollate [0.1 % (w/v), Sigma]. Growth at different temperatures (4, 10, 15, 18, 25, 28, 30, 35, 37 and 45 °C) and pH conditions (pH 4.0–10.0, at intervals of 0.5 pH units) were determined in R2A broth. Two different buffers were used (final concentration, 100 mM): acetate buffer was used for pH 4.0–6.5 and phosphate buffer was used for pH 7.0–10.0. Salt tolerance was tested in R2A broth containing 0–5 % (w/v) NaCl (at intervals of 0.5 %). Growth was estimated by monitoring the optical density at 600 nm. Oxidase and catalase activity were tested with 1 % (w/v) N, N, N0 , N0 -tetramethyl-1, 4-phenylenediamine reagent and 3 % (v/v) H2O2, respectively. Tests for hydrolysis of DNA (DNase agar, Oxoid), casein [on R2A agar supplemented with 2 % skim milk (Oxoid)], starch [on R2A agar containing 1 % starch (Difco)], carboxymethyl-cellulose (CMC) [on R2A agar containing 0.1 % CMC (Sigma)], L-tyrosine [on R2A agar containing 0.5 % L-tyrosine (Sigma)], chitin [on R2A agar containing 1 % chitin from crab shell (Sigma)], esculin [on R2A agar containing 0.1 % esculin and 0.02 % ferric citrate (Difco)], Tween 20 [on R2A agar containing 0.01 % CaCl22H2O and 1 % Tween 20 (Sigma)] and Tween 80 [on R2A agar containing 0.01 % CaCl22H2O and 1 % Tween 80 (Sigma)] were performed. Chemolithoautotrophic growth of strain THGSQE7T with hydrogen gas was tested on Medium 81 agar (http://www.dsmz.de/microorganisms/medium/ pdf/DSMZ_Medium81.pdf) under the conditions described by Malik and Schlegel (1981). In the above description, except specially indicated, all the tests were evaluated after 7 days of incubation at 28 °C. In addition, API 20NE and API ZYM tests (bioMe´rieux) were used for evaluating basic chemical test, carbon source assimilation and enzyme activity, according to the manufacturer’s instructions. Strains H. pseudoflava KCTC 2348T, H. bisanensis KCTC 12980T and H. flava KCTC 1648T were included as references for the investigation of the biochemical tests using the same laboratory conditions.
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Molecular characterization and phylogenetic construction
values were converted to percentage DNA–DNA relatedness values.
Genomic DNA was extracted and purified using a Genomic DNA extraction kit (Solgent, Korea). The 16S rRNA gene was amplified from the chromosomal DNA with the universal bacterial primer pair 27F and 1492R according to the methods of Weisburg et al. (1991). Sequencing of 16S rRNA gene was performed by Solgent Co. Ltd (Korea). Sequences of related taxa were obtained from GenBank database and EzTaxon-e server (http://www.ezbiocloud.net/eztaxon; Kim et al. 2012). Multiple alignments were performed using CLUSTAL_X (Thompson et al. 1997), gap editions were performed using BioEdit program (Hall 1999). The neighbor-joining (Saitou and Nei 1987), maximum-likelihood (Felsenstein 1981) and maximumparsimony (Fitch 1971) methods were used to construct phylogenetic trees in MEGA 6 program package (Kumar et al. 2008; Tamura et al. 2013). All the species of the genus Hydrogenophaga were included in the phylogenetic trees. The Kimura two-parameter model (Kimura 1983) was used to calculate the evolutionary distances. The bootstrap values were calculated based on 1000 replications (Felsenstein 1985).
Chemotaxonomic characterization
DNA G?C content and DNA–DNA hybridization Genomic DNA of strain THG-SQE7T and the three closest reference strains were extracted as described elsewhere (Moore and Dowhan 1995). Genomic DNA of strain THG-SQE7T and Escherichia coli strain B (Sigma-Aldrich D4889) was degraded enzymatically into nucleosides by nuclease P1 and alkaline phosphatase (Sigma). The DNA G?C content was analyzed using reverse-phase HPLC system (Alliance 2690 system, Waters) as described by Mesbah et al. (1989), the E. coli strain B was used as standard. DNA–DNA hybridization was performed between strain THG-SQE7T and H. pseudoflava KCTC 2348T, H. bisanensis KCTC 12980T and H. flava KCTC 1648T, using photobiotin-labelled probes in microplate wells (Ezaki et al. 1989). Optimum hybridization temperature was at 43 °C. Hybridization was performed with five replications for each sample. The highest and lowest values obtained for each sample were excluded and the means of the remaining three
For quinone and polar lipids analysis, cells were grown in R2A broth at 28 °C for 3 days, collected by centrifugation and freeze-dried. Isoprenoid quinones analysis of strain THG-SQE7T and its closest reference strain H. pseudoflava KCTC 2348T was performed using RP-HPLC Waters 2690 Alliance system as previously described (Collins and Jones 1981; Hiraishi et al. 1996). Polar lipids of strains THGSQE7T and H. pseudoflava KCTC 2348T were analyzed by using two-dimensional thin layer chromatography (Minnikin et al. 1984). The lipids were visualized using the following reagents: 5 % molybdatophosphoric acid (total lipids, Sigma), 0.2 % ninhydrin (aminolipids, Sigma), and 2.5 % anaphthol-sulfuric acid (glycolipids, Sigma) and molybdenum blue (phospholipids, Sigma). For fatty acid analysis, strain THG-SQE7T and the three closest reference strains were grown on R2A agar at 28 °C for 2 days (the type strains exhibited similar growth rates). The cellular fatty acid profiles were analysed according to the protocol of Sherlock Microbial Identification System (MIDI) and identified with GC (Hewlett Packard 6890) using Sherlock Aerobic Bacterial Database (TSBA60) (Sasser 1990).
Results and discussion Phenotypic analysis showed that cells of strain THGSQE7T were Gram-staining negative, strictly aerobic and rod-shaped, approximately 0.60–0.70 9 1.85–2.40 lm, and motile by means of a polar flagellum (Supplementary Fig. S2), which are typical characteristics for bacteria of the genus Hydrogenophaga (Willems et al. 1989). The comparative analysis of biochemical and physiological characteristics of strain THG-SQE7T and H. pseudoflava KCTC 2348T, H. bisanensis KCTC 12980T and H. flava KCTC 1648T indicated that all strains are motile, positive for nitrate reduction, negative for hydrolysis of starch, gelatin and chitin. All strains are positive for esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase
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activity; negative for cystine arylamidase, trypsin, achymotrypsin, a-galactosidase, b-glucuronidase, bgalactosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase activity. All strains are negative for indole production, glucose acidification, and assimilation of adipic acid, capric acid, citric acid and phenylacetic acid; positive for assimilation of D-glucose and gluconate. The distinction between strain THG-SQE7T and the three closely related reference strains were in production of lipase, urease, b-glucosidase, casein hydrolysis and Nacetyl-glucosamine; THG-SQE7T is unable to hydrolyse the DNA, in contrast to the other three strains (other details are given in Table 1). Phenotypic and biochemical properties suggested that stain THG-SQE7T is clearly distinguished form previously described species of the genus Hydrogenophaga and may represent a novel species. The phylogenetic analysis based on the 16S rRNA gene sequence of strain THG-SQE7T (NCBI accession number is KM598239) confirmed that strain THGSQE7T belongs to the genus Hydrogenophaga, and is most closely related to H. pseudoflava ATCC 33668T (98.4 %), followed by H. bisanensis K102T (97.6 %) and H. flava CCUG 1658T (97.6 %), while 16S rRNA gene sequence similarity with other species of the genus were found to be below 96.6 % (Fig. 1, Supplementary Fig. S1). According to genetic analysis, strain THGSQE7T showed 53.5, 36.0 and 22.5 % DNA–DNA relatedness with H. pseudoflava KCTC 2348T, H. bisanensis KCTC 12980T and H. flava KCTC 1648T, respectively. The DNA relatedness values between strain THG-SQE7T and three most phylogenetically closely related strains were found to be significantly lower than the recommended threshold of 70 % (Wayne et al. 1987; Stackebrandt and Goebel 1994). The DNA G?C content of the novel isolate was 63.7 mol%, which conforms to the expected range of G?C contents for the genus Hydrogenophaga. The polar lipid profiles of strain THG-SQE7T and H. pseudoflava KCTC 2348T are shown in Supplementary Fig. S3. The polar lipid profile of strain THGSQE7T was found to be different to that of H. pseudoflava KCTC 2348T by the presence of unidentified lipids (L4 and L5) and by the lack of unidentified phospholipid (PL1) and an unidentified lipid (L0). Strain THG-SQE7T contains Q-8 as the only respiratory quinone, which is a typical characteristic for bacteria of the genus Hydrogenophaga (Willems et al.
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Antonie van Leeuwenhoek (2015) 108:695–701 Table 1 Physiological characteristics of THG-SQE7T and the related species of the genus Hydrogenophaga Characteristics
1
2
3
4
Hydrolysis of: Esculin
?
?
-
-
L-Tyrosine
-
-
?
-
Casein
?
-
-
-
CMC
?
?
-
?
DNA
-
?
?
?
Tween 20
-
?
-
-
Tween 80
-
?
-
-
D-maltose
?
?
-
?
D-mannitol
?
?
-
-
Assimilation of
D-Mannose
?
?
-
-
L-Arabinose
?
?
-
-
Malic acid
?
?
-
?
?
-
-
-
Urease
?
-
-
-
Alkaline phosphatase
-
?
-
?
Lipase (C14)
W
-
-
-
Valine arylamidase
?
?
-
-
a-glucosidase
?
?
-
?
b-glucosidase
?
-
-
-
?
-
?
N-Acetyl-glucosamine Enzyme activities
Arginine dihydrolase T
T
Strains 1 THG-SQE7 , 2 H. pseudoflava KCTC 2348 , 3 H. bisanensis KCTC 12980T, 4 H. flava KCTC 2348T (type strain of the genus) All data was carried out in this study ? positive, - negative, w weakly positive
1989). The major fatty acids of strain THG-SQE7T were identified as summed feature 3 (C16:1x7c and/or C16:1x6c) (51.9 %), C16:0 (22.5 %) and C18:1x7c (12.2 %). The fatty acid composition of strain THG-SQE7T was very similar to H. pseudoflava KCTC 2348T, H. bisanensis KCTC 12980T and H. flava KCTC 2348T (Table 2). The phylogenetic analysis based on the 16S rRNA gene sequence similarity, phenotypic and chemotaxonomic properties revealed that strain THG-SQE7T can be clearly differentiated from other members of the genus Hydrogenophaga and represents a novel species, for which the name Hydrogenophaga luteola sp. nov. is proposed.
Antonie van Leeuwenhoek (2015) 108:695–701
98
699
Hydrogenophaga taeniospiralis ATCC 49743T (AF078768)
91
*Hydrogenophaga
carboriunda YZ2T (EU095331)
Hydrogenophaga palleronii DSM 63T (AF019073)
91
T 100 Hydrogenophaga atypica BSB 41.8 (AJ585992)
83
Hydrogenophaga defluvii BSB 9.5T (AJ585993) Hydrogenophaga ntermedia S1T (AF019037) Hydrogenophaga bisanensis K102T (EF532793)
74
Hydrogenophaga flava CCUG 1658T (AF078771) Hydrogenophaga luteola THG-SQE7T (KM598239)
83
92
86
Hydrogenophaga pseudoflava ATCC 33668T (AF078770) * Hydrogenophaga * Hydrogenophaga
83
97
temperata TR7-01T (AB166886)
electricum AR20T (AB746948)
Malikia granosa P1T (AJ627188) Malikia spinosa ATCC 14606T (AB021387) Macromonas bipunctata IAM 14880T (AB077037) Tepidicella xavieri TU-16T (DQ295805) Hydrogenophaga caeni EMB71T (DQ372983) Xenophilus aerolatus 5516S-2T (EF660342) Brachymonas chironomi AIMA4T (EU346912) Diaphorobacter oryzae RF3T (EU342381)
Delftia litopenaei wsw-7T (GU721027) Comamonas odontotermitis Dant 3-8T (DQ453128) Comamonas badia DSM 17552T (AXVM01000023) 0.005 Fig. 1 Neighbor-joining tree based on 16S rRNA gene sequence analysis, showing relationships between strain THGSQE7T and related members of the genus Hydrogenophaga. Bootstrap values (expressed as percentage of 1000 replications)
over 70 % are shown at the branching points. Comamonas badia DSM 17552T (AXVM01000023) was used as outgroup. Bar 0.005 substitutions per nucleotide position. Symbol * indicated the non-valid species of genus Hydrogenophaga
Description of Hydrogenophaga luteola sp. nov
not on LBA or MacConkey agar. Catalase and oxidase positive, able to hydrolyze esculin, CMC and casein, but not able to hydrolyze chitin, gelatin, starch, Tween 20, Tween 80 or L-tyrosine. Shows chemolithotrophic growth, using the oxidation of H2 as an energy source. Positive for esterase lipase (C8), esterase (C4), leucine arylamidase, valine arylamidase, acid phosphatase, aglucosidase, b-glucosidase and naphthol-AS-BI-phosphohydrolase; negative for alkaline phosphatase, agalactosidase, cystine arylamidase, trypsin, a-chymotrypsin, a-fucosidase, a-mannosidase, b-galactosidase, b-glucuronidase and N-acetyl-bglucosaminidase; weakly positive for lipase (C14).
Hydrogenophaga luteola (lu.te’o.la. L. masc. adj. luteola yellowish, referring to the colour of the colonies on R2A agar) Cells are Gram-staining negative, strictly aerobic, motile by means of a polar flagellum and rod-shaped (0.60–0.70 9 1.85–2.40 lm). Colonies are round, yellowish, non-sticky, translucent and convex with entire margins on R2A agar. Growth occurs at 18–37 °C (optimum, 28–37 °C); at pH 6.0–9.0 (optimum, 6.5–8.0) and at 0–2.0 % NaCl (optimum, 0–0.5 %). Grows on R2A agar, NA and TSA, but do
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Table 2 Cellular fatty acid profiles of THG-SQE7T and the related species of the genus Hydrogenophaga Fatty acid
1
2
3
4
4.2
Straight C14:0
4.3
3.5
Tr
C16:0
22.5
25.4
23.3
23.1
C17:0
Tr
1.8
1.0
1.6
C18:0
Tr
Tr
2.7
Tr
Unsaturated C17:1 x6c
Tr
1.2
2.0
1.6
C18:1 x7c
12.2
15.6
17.7
18.9
C19:1 x6c
ND
ND
3.3
ND
Tr
ND
1.0
ND
C10:0 3OH
4.5
3.5
ND
3.6
C18:0 3OH
ND
ND
Tr
ND
51.9
45.8
42.4
43.4
Branched-chain Anteiso-C12:0 Hydroxy
Summed feature 3a
Strains 1 THG-SQE7T, 2 H. pseudoflava KCTC 2348T, 3 H. bisanensis KCTC 12980T, 4 H. flava KCTC 2348T All data was carried out in this study Fatty acids of less than 0.5 % in all strains were not listed ND not detected, Tr traces (\1.0 %) Summed feature 3a consisted of C16:1x7c/C16:1x6c
The results of indole production, glucose acidification, protease activity and assimilation of adipic acid, capric acid, phenylacetic acid and citric acid are negative; the results of nitrate reduction, b-glucosidase, arginine dihydrolase, urease activity, D-maltose, D-mannitol, D-glucose, L-arabinose, D-mannose, Nacetyl-glucosamine, gluconate and malic acid assimilation are positive. The polar lipid profiles consist of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and five unidentified lipids. The major fatty acids are summed feature 3 (C16:1x7c and/or C16:1x6c), C16:0 and C18:1x7c. Contains Q-8 as the only respiratory quinone. The DNA G?C content of the type strain is 63.7 mol%. The type strain, THG-SQE7T (=KCTC 42501T = CCTCC AB 2014314T = JCM 30433T), was isolated from reed pond water in Shangqiu, PR China. Acknowledgments This work was conducted under the industrial infrastructure program (No. N0000888) for fundamental technologies which is funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea).
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