Antonie van Leeuwenhoek (2014) 105:245–251 DOI 10.1007/s10482-013-0071-6
ORIGINAL PAPER
Parvularcula oceanus sp. nov., isolated from deep-sea water of the Southeastern Pacific Ocean Shuhui Li • Kai Tang • Keshao Liu Chang-Ping Yu • Nianzhi Jiao
•
Received: 13 September 2013 / Accepted: 29 October 2013 / Published online: 7 November 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract A Gram-negative, aerobic, motile by single polar flagellum, short rod-shaped bacterium was isolated from a deep-seawater sample of the Southeastern Pacific Ocean. Growth was found to occur at 10–40 °C, at pH 4.0–10.0 and in the presence of 0–9.0 % (w/v) NaCl. The phylogenetic tree based on 16S rRNA gene sequences showed that strain JLT2013T fell within a clade comprising species of the genus Parvularcula and formed a coherent cluster with Parvularcula lutaonensis CC-MMS-1T (neighbour-joining phylogenetic tree) or Parvularcula dongshanensis SH25T (maximumlikelihood and maximum-parsimony phylogenetic trees). Sequence similarity analyses based on 16S rRNA gene sequences revealed that strain JLT2013T shows high sequence similarity to P. lutaonensis CCMMS-1T (96.7 %), P. dongshanensis SH25T (96.0 %) and Parvularcula bermudensis HTCC2503T (95.2 %). The major cellular fatty acids were identified as C12:0
(34.3 %), summed feature 8 (C18:1x7c and/or C18:1x6c) (10.9 %), C16:0 (10.0 %) and C17:1x6c (7.2 %). The polar lipids were found to include diphosphatidylglycerol, phosphatidylglycerol, three sphingoglycolipids and three unknown glycolipids. Strain JLT2013T was found to contain Q-10 as the predominant quinone. The DNA G?C content was determined to be 66.3 mol%. In the light of the phenotypic characteristics, chemotaxonomic features and phylogenetic evidence gathered in this study, strain JLT2013T (=LMG 27362T = CGMCC 1.12400T) should be classified as a novel species in the genus Parvularcula, for which the name Parvularcula oceanus sp. nov. is proposed. Keywords Parvularcula oceanus sp. nov. Aerobic Deep-sea water
Introduction Electronic supplementary material The online version of this article (doi:10.1007/s10482-013-0071-6) contains supplementary material, which is available to authorized users. S. Li K. Tang (&) K. Liu N. Jiao State Key Laboratory for Marine Environmental Science, Institute of Marine Microbes and Ecospheres, Xiamen University, Xiamen 361005, People’s Republic of China e-mail: [email protected] C.-P. Yu Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, People’s Republic of China
The genus Parvularcula was first described by Cho and Giovannoni (2003). Parvularcula bermudensis, the first member of the genus, was characterized as being Gram-negative, strictly aerobic, chemoheterotrophic, orange, slightly motile short rods with a single flagellum, which was isolated from the Bermuda Atlantic Time Series Station in the western Sargasso Sea, Atlantic Ocean (Cho and Giovannoni 2003). Subsequently, Parvularcula lutaonensis, another species in the genus, was isolated from a coastal hot spring at Lutao on a small volcanic island off the east
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coast of Taiwan by Arun et al. (2009). Recently, Parvularcula dongshanensis was isolated from soft coral collected from Dongshan Island, China (Yu et al. 2013). In this work, on the basis of polyphasic taxonomic characterization, we propose that strain JLT2013T represents a novel species within the genus Parvularcula, for which the name Parvularcula oceanus sp. nov. is proposed.
Materials and methods Isolation and cultivation of strains A deep-sea water sample (at a depth of 800 m) was collected from the Southeastern Pacific Ocean. Strain JLT2013T was isolated using a standard dilution plating method culturing technique on marine agar 2216 (MA; BD) at 30 °C. Seawater sample (200 ll) was taken and spread on MA plates, which were then incubated at 30 °C for 3 days. Strain JLT2013T was isolated and subsequently purified on MA at 30 °C for 2 weeks. The culture was routinely grown in marine broth 2216 (MB; BD) and was preserved as glycerol suspensions (15 %, v/v) at -80 °C. The reference strains P. lutaonensis CC-MMS-1T and P. bermudensis HTCC2503T were obtained from Korean Collection for Type Cultures. P. dongshanensis SH25T was obtained from Marine Culture Collection of China. They were cultured under identical conditions as strain JLT2013T for comparison. Phenotypic and chemical characterization Colony morphology and pigmentation were observed from cultures grown aerobically on MA after 2 days at 25 °C. Cell size and morphology were observed by using transmission electron microscopy (JEM-1230; JEOL USA), using cells grown on MA for 2 days at 25 °C. Unless specified otherwise, physiological characteristics were examined at 25 °C using MA as the basal medium. Gram-staining was performed using the method described by Gerhardt et al. (1994). Growth at different temperatures was tested at 4, 16, 20, 25, 30, 35 and 40 °C in MB with pH 7.8, 2.0 % NaCl. The pH range for optimal growth was determined in MB adjusted to pH 4.0–10.0 (at intervals of 1.0 pH units). Salinity requirements were tested using MB medium with final NaCl concentrations of 0, 0.5,
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and 1.0–12.0 %, at intervals of 1.0 % (w/v) (at pH 7.8 and 25 °C). Hydrolysis of casein, starch and DNA were determined according to the method of Smibert and Krieg (1994). Accumulation of poly-b-hydroxybutyrate (PHB) granules in cells was observed by Nile blue A staining (Ostle and Holt 1982). The presence of bacteriochlorophyll a was tested according to the method of Pukall et al. (1999). Other basic physiological and biochemical tests were performed using API 20E, API 20NE and API ZYM strips (bioMe´rieux) according to the manufacturers’ instructions. Carbon source utilization was determined using Biolog GN2 microplates as described by Ru¨ger and Krambeck (1994). Susceptibility to antimicrobial agent was performed using the disc-diffusion plate method (Fraser and Jorgensen 1997) with discs containing ampicillin (10 lg), carbenicillin (100 lg), chloromycetin (5 lg), erythromycin (15 lg), gentamicin (10 lg), kanamycin (30 lg), lincomycin (2 lg), neomycin (30 lg), novobiocin (5 lg), penicillin (10 lg), polymyxin B (300 lg), rifampicin (5 lg), streptomycin (10 lg), tetracycline (30 lg), vancomycin (30 lg). Bacterial cultures (200 ll) were spread on MA plates, discs impregnated with antibiotics were placed on the plate surfaces, and the plates were incubated for 3 days at 25 °C. Chemotaxonomy The DNA G?C content of strain JLT2013T was estimated by the HPLC method (Mesbah et al. 1989) after DNA extraction and purification according to the method of Marmur (1961). Isoprenoid quinones were extracted from 100 mg of freeze dried cell material, which was obtained from cells grown on MB at 25 °C for 3 days. Quinones were extracted using the twostage method described by Tindall (1990a, b) and analysed by HPLC. Polar lipids were extracted and analysed by two-dimensional thin-layer chromatography (Collins et al. 1980; Kates 1986), using Merck silica ge1 60F254 plates (10 by 20 cm) and chloroform–methanol–water (65:25:4, vol/vol) in the first dimension and chloroform–methanol–acetic acid– water (80:12:15:4, vol/vol) in the second dimension. The general detection reagent, sulfuric acid–ethanol (1:2, vol/vol), was used to detect total polar lipids. For cellular fatty acid analysis, cells were grown on MA at 25 °C for 2 days and analysed according to the
Antonie van Leeuwenhoek (2014) 105:245–251
method described by Sasser (1990) using MIDI Sherlock version 6.0 and the TSBA6 6.00 library.
16S rRNA gene analysis and PCR amplification Genomic DNA was extracted using a TIANamp Bacteria DNA Kit (Tiangen Biotech, DP302) with cells grown in MB medium for 2 days at 30 °C and subsequently washed and resuspended in TE buffer (Zheng 2010). The DNA yield and concentration were assessed using by the A280/ A260 and A230/A260 ratio values with a NANODROP 2000 spectrophotometer (Thermo Scientific, USA). The 16S rRNA gene of strain JLT2013T was amplified using universal bacterial primers 27F (50 -AGAGTTTGA TCCTGGCTCAG-30 ) and 1492R (50 -GGTTACCTTGTT ACGACTT-30 ) (Embley 1991). The nearly complete 16S rRNA gene sequence of strain JLT2013T was submitted to the GenBank database (NCBI) and compared with those available using the BLAST program (http://blast.ncbi.nlm. nih.gov/Blast.cgi, NCBI). Based on 16S rRNA gene sequences, phylogenetic analyses (with neighbour-joining, maximum likelihood and maximum parsimony
247 Table 1 Differential phenotypic characteristics between strain JLT2013T and type strains of other species of the genus Parvularcula Characteristic
1
2
3
4
Colony colour
Orange
Orange
Orange
Yellowish brown
Optimum temperature (°C) Oxidase
30
25–30
25–40
ND
?
?
-
ND
Catalase
?
-
?
ND
Urea
-
?
?
-
Aesculin
?
?
?
-
Gelatin
-
-
-
?
Hydrolysis of
Enzymic activity (API ZYM) a–Chymotrypsine
-
?
?
-
Cystine arylamidase
-
-
?
-
Esterase (C4)
?
-
?
?
Esterase lipase (C8)
?
?
-
?
a-Galactosidase
-
-
?
-
b-Galactosidase
-
?
-
-
a-Glucosidase
?
-
-
-
b-Glucosidase
?
?
?
-
b-Glucuronidase
-
-
?
-
Phosphatase acide
?
-
?
-
Trypsine
-
?
-
-
Valine arylamidase
?
-
-
-
Acid production from (API 20E) Melibiose
?
-
-
-
Arabinose
?
-
?
-
-
?
-
-
-
-
?
Voges–Proskauer test
Assimilation of (API 20NE) N-Acetylglucosamine
Fig. 1 Transmission electron micrograph of strain JLT2013T cells negatively stained with 1 % (w/v) phosphotungstic acid. Bar, 1 lm
-
Adipic acid
-
-
-
?
L-Arabinose
-
-
?
?
L-Arginine
-
?
?
-
D-Glucose
-
?
?
?
D-Mannose
-
-
-
?
D-Maltose
-
?
-
-
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Antonie van Leeuwenhoek (2014) 105:245–251
Table 1 continued Characteristic
1
2
3
4
Potassium gluconate
-
-
-
?
Table 2 Fatty acid compositions (%) of members of the genus Parvularcula Fatty acid
1
2
3
4
-
1.3
Straight-chain:
Strains: 1. JLT2013T; 2. Parvularcula lutaonensis CC-MMS1T; 3. Parvularcula dongshanensis SH25T; 4. Parvularcula bermudensis HTCC2503T
C10:00
10.8
-
C12:00
34.0
8.7
C14:00
-
-
-
4.4
All data were obtained from this study for Parvularcula lutaonensis CC-MMS-1T, Parvularcula dongshanensis SH25T and Parvularcula bermudensis HTCC2503T in parallel tests with strain JL2013T under identical conditions
C16:0 alcohol
1.6
-
-
-
C16:00
10.0
24.1
17.4
23.8
C17:00
-
-
-
1.1
? Positive;- negative, ND no data available
C18:00
7.3
6.0
16.6
26.8
iso-C10:0
2.5
-
-
-
anteiso-C14:0
3.9
-
-
-
anteiso-C15:0
2.2
-
-
-
anteiso-C17:0
3.5
-
-
tr
iso-C20:0
-
-
-
14.0
4.4
-
-
-
1.7
-
-
3.1
8.5
Branched saturated acids:
algorithms) were carried out using the method described by Kim et al. (1998). The 16S rRNA gene sequence of JLT2013T was aligned manually with sequences for representatives of the class Alphaproteobacteria derived from GenBank databases using the AL16S program (Chun 1995). The identification of phylogenetic neighbours was achieved using the EzTaxon-e (Kim et al. 2012). Phylogenetic trees were constructed within MEGA version 5 (Tamura et al. 2011) after sequence alignment using the ClustalW algorithm embedded into Bioedit (Hall 1999).
Unsaturated acids: C14:1x5c C18:1x9c
Branched unsaturated acids: C14:0 2-OH
4.0
8.7
-
-
C15:0 3-OH
1.5
-
-
-
cyclo-C19:0x8c
-
-
5.7
-
10.9
52.4
60.4
16.2
Summed featuresa
Results and discussion Phenotypic characteristics Colonies of strain JLT2013T were observed to be orange, opaque, smooth, circular, convex and 0.7–1.1 mm in diameter after cultivation on MA at 25 °C for 3 days. The strain was observed to be Gram negative, motile by single polar flagellum, short rods, 1.5–2.3 lm long and 0.8–1.5 lm wide (Fig. 1). The temperature range for growth was found to be 10–40 °C with an optimum of 30 °C. Growth was found to occur at pH 4.0–10.0 (optimum 5.0–9.0) and at 0–9.0 % NaCl (optimum 3.0–6.0 %). The catalase and oxidase activities were found to be positive. Strain JLT2013T was found to hydrolyse aesculin and Tweens 40 and 80. The Voges–Proskauer reaction, nitrate reduction, indole and H2S production were negative. Strain JLT2013T and Parvularcula reference type strains were found to be positive for activity of alkaline and acid phosphatase, leucine arylamidase, naphtol-AS-BI- phosphohydrolase. Furthermore, strain JLT2013T and Parvularcula
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Summed Feature 8
Values are percentages of total fatty acids Strains: 1. JLT2013T; 2. Parvularcula lutaonensis CC-MMS1T; 3. Parvularcula dongshanensis SH25T; 4. Parvularcula bermudensis HTCC2503T All data were obtained from this study for Parvularcula lutaonensis CC-MMS-1T, Parvularcula dongshanensis SH25T and Parvularcula bermudensis HTCC2503T in parallel tests with strain JL2013T under identical conditions - not detected, tr values are less than 1.0 % a
Summed features are groups of two or three fatty acids that cannot be separated by GLC with the MIDI System. Summed feature 8, C18:1x7c and/or C18:1x6c
reference type strains were found to be negative for hydrolysis of casein, starch, and gelatin; nitrate reduction and H2S production; assimilation of Dmannose, capric acid, malic acid, trisodium citrate and phenylacetic acid; acid production from glucose, mannitol, inositol, sorbitol, rhamnose, sucrose and amygdalin; and activity of lipase (C14), N-acetyl-bglucosaminidase, a-mannosidase and b–fucosidase. However, only strain JLT2013T was found to be
Antonie van Leeuwenhoek (2014) 105:245–251
249
84 63 93 92 92 90 100
100 100 65
Mesorhizobium australicum WSM2073T (AY601516) Nitratireductor basaltis J3T (EU143347) Shinella granuli Ch06T (AY995149) Afifella pfennigii DSM17143T (FR733717) Lutibaculum baratangense AMV1T (FN297835) Parvibaculum indicum P31T (FJ182044) Parvibaculum lavamentivorans DS-1T (CP000774) Parvularcula bermudensis HTCC2503T (AF544015) Parvularcula dongshanensis SH25T (JQ778314) Parvularcula oceanus JLT2013T (JX845635) Parvularcula lutaonensis CC-MMS-1T (EU346850) Hellea balneolensis 26III/A02/215T (AY576758) Rickettsia prowazekii ATCC VR-142T (M21789)
0.02
Fig. 2 Neighbour-joining phylogenetic tree showing the relationships between strain JLT2013T and representatives of the class Alphaproteobacteria based on 16S rRNA gene sequences. Only bootstrap values above 50 % are shown (1,000 replications). Rickettsia prowazekii ATCC VR-142T was used as an
outgroup to define the root of the tree. Bar, 0.02 estimated sequence divergence. Filled circles indicate that the corresponding nodes were also recovered in the trees generated with the maximum-likelihood and maximum-parsimony algorithms
positive for acid production from melibiose and activity of valine arylamidase and a-glucosidase. The reference type strains of other Parvularcula species were found to be able to utilize D-glucose whereas strain JLT2013T could not. More detailed results of the physiological characterisations are given in Table 1. Strain JLT2013T showed sensitivity to kanamycin, gentamicin, rifampicin, neomycin and streptomycin. The DNA G?C content of strain JLT2013T was determined to be 66.3 mol%. The polar lipids of strain JLT2013T was identified as diphosphatidylglycerol, phosphatidylglycerol, three sphingoglycolipids and three unknown glycolipids (Fig. S1). The fatty acid profile determined for the novel isolate differed from those of recognized species of the genus Parvularcula (Cho and Giovannoni 2003; Arun et al. 2009; Yu et al. 2013) in the proportions of C16:0, C18:0 and summed feature 8 (C18:1x7c and/or C18:1x6c). Furthermore, C16:0 alcohol, iso-C10:0, anteiso-C14:0, anteiso-C15:0, C14:1x5c and C15:0 3-OH were detected only in cells of strains JLT2013T, whereas C14:0, C17:0 and isoC20:0 were found only in cells of P. dongshanensis SH25T and cyclo-C19:0x8c only in cells of P. bermudensis HTCC2503T (Table 2). Like other species of the genus Parvularcula, the predominant respiratory quinone in strain JLT2013T was found to be Q-10.
Phylogenetic analysis A nearly complete 16S rRNA gene sequence of strain JLT2013T (1426 bp, GenBank accession number: JX845635) was obtained. Phylogenetic distances and clustering obtained via the neighbor-joining (Saitou and Nei 1987) are shown in Fig. 2. As the phylogenetic tree shows, strain JLT2013T fell within a clade comprising species of the genus Parvularcula and formed a coherent cluster with P. lutaonensis CC-MMS-1T. A similar result was observed in the maximum-likelihood and maximum-parsimony tree (Fig. S2, S3), although in these trees strain clustered with P. dongshanensis SH25T. Strain JLT2013T exhibited 96.7 %, 96.0 % and 95.2 % 16S rRNA gene sequence similarity with P. lutaonensis CC-MMS-1T, P. dongshanensis SH25T and P. bermudensis HTCC2503T, respectively. Taxonomic conclusion Based on 16S rRNA gene sequence analysis, strain JLT2013T exhibits high 16S rRNA gene sequence similarity with P. lutaonensis CC-MMS-1T (96.7 %), P. dongshanensis SH25T (96.0 %) and P. bermudensis HTCC2503T (95.2 %). The phylogenetic association of strain JLT2013T with the type strains of members of the genus Parvularcula is consistent as indicated by neighbour-joining, maximum likelihood and
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maximum-parsimony trees (Fig. 1; Fig. S2, S3), although variations in topology are noted. Strain JLT2013T is distinguishable from the members of the genus Parvularcula by differences in polyphasic taxonomic characterization, such as hydrolysis of aesculin and urea, enzymic activity of valine arylamidase, a-glucosidase and melibiose, DNA G?C content and fatty acid composition. Based on phylogenetic analysis and phenotypic characteristics, strain JLT2013T can be considered to represent a novel species of the genus Parvularcula, for which the name P. oceanus sp. nov. is proposed.
phosphatidylglycerol, three sphingoglycolipids and three unknown glycolipids. The predominant cellular fatty acids are C12:0, summed feature 8 as defined by the MIDI system (C18:1x7c and/or C18:1x6c), C10:0, C16:0, and C18:0. The predominant respiratory quinone is Q-10. The DNA G ? C content of the type strain is 66.3 mol%. The type strain is JLT2013T (=LMG 27362T = CGMCC 1.12400T), isolated from deep-sea water in Southeastern Pacific. The GenBank accession number for the 16S rRNA gene sequence of strain JLT2013T is JX845635.
Description of P. oceanus sp. nov.
Acknowledgements This work was supported by the National Key Basic Research Program of China (2013CB955700), the SOA Project (201105021), the National Natural Science Foundation of China project (41276131, 91028001) and MELRI1304.
P. oceanus (o.cean.us. N.L. fem. n. oceanus, the ocean, a bacterium isolated from a marine environment) Cells are Gram-stain negative, aerobic, motile by single polar flagellum, short rods, (1.5–2.3 lm in length and 0.8–1.5 lm in diameter). Colonies incubation on MA are circular, smooth, convex, orangecoloured. Bacterial growth is observed at temperature 10–40 °C (optimum, 30 °C), at pH 4.0–10.0 (optimum, 5.0–9.0) and with 0–9.0 % (w/v) NaCl (optimum, 3.0–6.0 % NaCl). Positive for oxidase and catalase. Poly-b-hydroxybutyrate granules are produced and bacteriochlorophyll a is absent. Aesculin, Tweens 40 and 80 are hydrolysed but gelatin, starch, casein, urea and DNA are not. Nitrate is not reduced. Indole and H2S are not produced. Negative for Voges– Proskauer reaction. According to the API 20E tests, acid is formed from melibiose but not from glucose, mannitol, inositol, sorbitol, rhamnose, sucrose or amygdalin. In assays with the API ZYM system, alcaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, aglucosidase and b-glucosidase are present but lipase (C14), cystine arylamidase, trypsine, a-chymotrypsine, a-galactosidase, b-galactosidase, b-glucuronidase N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase are absent. The following substrates are oxidized as carbon sources in Biolog GN2 tests: Tween-40, Tween-80, adonitol, methyl pyruvate, formic acid, D-galactonic acid lactone, D-gluconicacid, p-hydroxy phenylacetic acid, a-ketovaleric acid, malonic acid, sebacic acid, succinamic acid, glycyl-Lglutamic acid, urocanic acid and glycerol. The polar lipids consist of diphosphatidylglycerol,
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ORIGINAL PAPER
Parvularcula oceanus sp. nov., isolated from deep-sea water of the Southeastern Pacific Ocean Shuhui Li • Kai Tang • Keshao Liu Chang-Ping Yu • Nianzhi Jiao
•
Received: 13 September 2013 / Accepted: 29 October 2013 / Published online: 7 November 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract A Gram-negative, aerobic, motile by single polar flagellum, short rod-shaped bacterium was isolated from a deep-seawater sample of the Southeastern Pacific Ocean. Growth was found to occur at 10–40 °C, at pH 4.0–10.0 and in the presence of 0–9.0 % (w/v) NaCl. The phylogenetic tree based on 16S rRNA gene sequences showed that strain JLT2013T fell within a clade comprising species of the genus Parvularcula and formed a coherent cluster with Parvularcula lutaonensis CC-MMS-1T (neighbour-joining phylogenetic tree) or Parvularcula dongshanensis SH25T (maximumlikelihood and maximum-parsimony phylogenetic trees). Sequence similarity analyses based on 16S rRNA gene sequences revealed that strain JLT2013T shows high sequence similarity to P. lutaonensis CCMMS-1T (96.7 %), P. dongshanensis SH25T (96.0 %) and Parvularcula bermudensis HTCC2503T (95.2 %). The major cellular fatty acids were identified as C12:0
(34.3 %), summed feature 8 (C18:1x7c and/or C18:1x6c) (10.9 %), C16:0 (10.0 %) and C17:1x6c (7.2 %). The polar lipids were found to include diphosphatidylglycerol, phosphatidylglycerol, three sphingoglycolipids and three unknown glycolipids. Strain JLT2013T was found to contain Q-10 as the predominant quinone. The DNA G?C content was determined to be 66.3 mol%. In the light of the phenotypic characteristics, chemotaxonomic features and phylogenetic evidence gathered in this study, strain JLT2013T (=LMG 27362T = CGMCC 1.12400T) should be classified as a novel species in the genus Parvularcula, for which the name Parvularcula oceanus sp. nov. is proposed. Keywords Parvularcula oceanus sp. nov. Aerobic Deep-sea water
Introduction Electronic supplementary material The online version of this article (doi:10.1007/s10482-013-0071-6) contains supplementary material, which is available to authorized users. S. Li K. Tang (&) K. Liu N. Jiao State Key Laboratory for Marine Environmental Science, Institute of Marine Microbes and Ecospheres, Xiamen University, Xiamen 361005, People’s Republic of China e-mail: [email protected] C.-P. Yu Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, People’s Republic of China
The genus Parvularcula was first described by Cho and Giovannoni (2003). Parvularcula bermudensis, the first member of the genus, was characterized as being Gram-negative, strictly aerobic, chemoheterotrophic, orange, slightly motile short rods with a single flagellum, which was isolated from the Bermuda Atlantic Time Series Station in the western Sargasso Sea, Atlantic Ocean (Cho and Giovannoni 2003). Subsequently, Parvularcula lutaonensis, another species in the genus, was isolated from a coastal hot spring at Lutao on a small volcanic island off the east
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coast of Taiwan by Arun et al. (2009). Recently, Parvularcula dongshanensis was isolated from soft coral collected from Dongshan Island, China (Yu et al. 2013). In this work, on the basis of polyphasic taxonomic characterization, we propose that strain JLT2013T represents a novel species within the genus Parvularcula, for which the name Parvularcula oceanus sp. nov. is proposed.
Materials and methods Isolation and cultivation of strains A deep-sea water sample (at a depth of 800 m) was collected from the Southeastern Pacific Ocean. Strain JLT2013T was isolated using a standard dilution plating method culturing technique on marine agar 2216 (MA; BD) at 30 °C. Seawater sample (200 ll) was taken and spread on MA plates, which were then incubated at 30 °C for 3 days. Strain JLT2013T was isolated and subsequently purified on MA at 30 °C for 2 weeks. The culture was routinely grown in marine broth 2216 (MB; BD) and was preserved as glycerol suspensions (15 %, v/v) at -80 °C. The reference strains P. lutaonensis CC-MMS-1T and P. bermudensis HTCC2503T were obtained from Korean Collection for Type Cultures. P. dongshanensis SH25T was obtained from Marine Culture Collection of China. They were cultured under identical conditions as strain JLT2013T for comparison. Phenotypic and chemical characterization Colony morphology and pigmentation were observed from cultures grown aerobically on MA after 2 days at 25 °C. Cell size and morphology were observed by using transmission electron microscopy (JEM-1230; JEOL USA), using cells grown on MA for 2 days at 25 °C. Unless specified otherwise, physiological characteristics were examined at 25 °C using MA as the basal medium. Gram-staining was performed using the method described by Gerhardt et al. (1994). Growth at different temperatures was tested at 4, 16, 20, 25, 30, 35 and 40 °C in MB with pH 7.8, 2.0 % NaCl. The pH range for optimal growth was determined in MB adjusted to pH 4.0–10.0 (at intervals of 1.0 pH units). Salinity requirements were tested using MB medium with final NaCl concentrations of 0, 0.5,
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and 1.0–12.0 %, at intervals of 1.0 % (w/v) (at pH 7.8 and 25 °C). Hydrolysis of casein, starch and DNA were determined according to the method of Smibert and Krieg (1994). Accumulation of poly-b-hydroxybutyrate (PHB) granules in cells was observed by Nile blue A staining (Ostle and Holt 1982). The presence of bacteriochlorophyll a was tested according to the method of Pukall et al. (1999). Other basic physiological and biochemical tests were performed using API 20E, API 20NE and API ZYM strips (bioMe´rieux) according to the manufacturers’ instructions. Carbon source utilization was determined using Biolog GN2 microplates as described by Ru¨ger and Krambeck (1994). Susceptibility to antimicrobial agent was performed using the disc-diffusion plate method (Fraser and Jorgensen 1997) with discs containing ampicillin (10 lg), carbenicillin (100 lg), chloromycetin (5 lg), erythromycin (15 lg), gentamicin (10 lg), kanamycin (30 lg), lincomycin (2 lg), neomycin (30 lg), novobiocin (5 lg), penicillin (10 lg), polymyxin B (300 lg), rifampicin (5 lg), streptomycin (10 lg), tetracycline (30 lg), vancomycin (30 lg). Bacterial cultures (200 ll) were spread on MA plates, discs impregnated with antibiotics were placed on the plate surfaces, and the plates were incubated for 3 days at 25 °C. Chemotaxonomy The DNA G?C content of strain JLT2013T was estimated by the HPLC method (Mesbah et al. 1989) after DNA extraction and purification according to the method of Marmur (1961). Isoprenoid quinones were extracted from 100 mg of freeze dried cell material, which was obtained from cells grown on MB at 25 °C for 3 days. Quinones were extracted using the twostage method described by Tindall (1990a, b) and analysed by HPLC. Polar lipids were extracted and analysed by two-dimensional thin-layer chromatography (Collins et al. 1980; Kates 1986), using Merck silica ge1 60F254 plates (10 by 20 cm) and chloroform–methanol–water (65:25:4, vol/vol) in the first dimension and chloroform–methanol–acetic acid– water (80:12:15:4, vol/vol) in the second dimension. The general detection reagent, sulfuric acid–ethanol (1:2, vol/vol), was used to detect total polar lipids. For cellular fatty acid analysis, cells were grown on MA at 25 °C for 2 days and analysed according to the
Antonie van Leeuwenhoek (2014) 105:245–251
method described by Sasser (1990) using MIDI Sherlock version 6.0 and the TSBA6 6.00 library.
16S rRNA gene analysis and PCR amplification Genomic DNA was extracted using a TIANamp Bacteria DNA Kit (Tiangen Biotech, DP302) with cells grown in MB medium for 2 days at 30 °C and subsequently washed and resuspended in TE buffer (Zheng 2010). The DNA yield and concentration were assessed using by the A280/ A260 and A230/A260 ratio values with a NANODROP 2000 spectrophotometer (Thermo Scientific, USA). The 16S rRNA gene of strain JLT2013T was amplified using universal bacterial primers 27F (50 -AGAGTTTGA TCCTGGCTCAG-30 ) and 1492R (50 -GGTTACCTTGTT ACGACTT-30 ) (Embley 1991). The nearly complete 16S rRNA gene sequence of strain JLT2013T was submitted to the GenBank database (NCBI) and compared with those available using the BLAST program (http://blast.ncbi.nlm. nih.gov/Blast.cgi, NCBI). Based on 16S rRNA gene sequences, phylogenetic analyses (with neighbour-joining, maximum likelihood and maximum parsimony
247 Table 1 Differential phenotypic characteristics between strain JLT2013T and type strains of other species of the genus Parvularcula Characteristic
1
2
3
4
Colony colour
Orange
Orange
Orange
Yellowish brown
Optimum temperature (°C) Oxidase
30
25–30
25–40
ND
?
?
-
ND
Catalase
?
-
?
ND
Urea
-
?
?
-
Aesculin
?
?
?
-
Gelatin
-
-
-
?
Hydrolysis of
Enzymic activity (API ZYM) a–Chymotrypsine
-
?
?
-
Cystine arylamidase
-
-
?
-
Esterase (C4)
?
-
?
?
Esterase lipase (C8)
?
?
-
?
a-Galactosidase
-
-
?
-
b-Galactosidase
-
?
-
-
a-Glucosidase
?
-
-
-
b-Glucosidase
?
?
?
-
b-Glucuronidase
-
-
?
-
Phosphatase acide
?
-
?
-
Trypsine
-
?
-
-
Valine arylamidase
?
-
-
-
Acid production from (API 20E) Melibiose
?
-
-
-
Arabinose
?
-
?
-
-
?
-
-
-
-
?
Voges–Proskauer test
Assimilation of (API 20NE) N-Acetylglucosamine
Fig. 1 Transmission electron micrograph of strain JLT2013T cells negatively stained with 1 % (w/v) phosphotungstic acid. Bar, 1 lm
-
Adipic acid
-
-
-
?
L-Arabinose
-
-
?
?
L-Arginine
-
?
?
-
D-Glucose
-
?
?
?
D-Mannose
-
-
-
?
D-Maltose
-
?
-
-
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Table 1 continued Characteristic
1
2
3
4
Potassium gluconate
-
-
-
?
Table 2 Fatty acid compositions (%) of members of the genus Parvularcula Fatty acid
1
2
3
4
-
1.3
Straight-chain:
Strains: 1. JLT2013T; 2. Parvularcula lutaonensis CC-MMS1T; 3. Parvularcula dongshanensis SH25T; 4. Parvularcula bermudensis HTCC2503T
C10:00
10.8
-
C12:00
34.0
8.7
C14:00
-
-
-
4.4
All data were obtained from this study for Parvularcula lutaonensis CC-MMS-1T, Parvularcula dongshanensis SH25T and Parvularcula bermudensis HTCC2503T in parallel tests with strain JL2013T under identical conditions
C16:0 alcohol
1.6
-
-
-
C16:00
10.0
24.1
17.4
23.8
C17:00
-
-
-
1.1
? Positive;- negative, ND no data available
C18:00
7.3
6.0
16.6
26.8
iso-C10:0
2.5
-
-
-
anteiso-C14:0
3.9
-
-
-
anteiso-C15:0
2.2
-
-
-
anteiso-C17:0
3.5
-
-
tr
iso-C20:0
-
-
-
14.0
4.4
-
-
-
1.7
-
-
3.1
8.5
Branched saturated acids:
algorithms) were carried out using the method described by Kim et al. (1998). The 16S rRNA gene sequence of JLT2013T was aligned manually with sequences for representatives of the class Alphaproteobacteria derived from GenBank databases using the AL16S program (Chun 1995). The identification of phylogenetic neighbours was achieved using the EzTaxon-e (Kim et al. 2012). Phylogenetic trees were constructed within MEGA version 5 (Tamura et al. 2011) after sequence alignment using the ClustalW algorithm embedded into Bioedit (Hall 1999).
Unsaturated acids: C14:1x5c C18:1x9c
Branched unsaturated acids: C14:0 2-OH
4.0
8.7
-
-
C15:0 3-OH
1.5
-
-
-
cyclo-C19:0x8c
-
-
5.7
-
10.9
52.4
60.4
16.2
Summed featuresa
Results and discussion Phenotypic characteristics Colonies of strain JLT2013T were observed to be orange, opaque, smooth, circular, convex and 0.7–1.1 mm in diameter after cultivation on MA at 25 °C for 3 days. The strain was observed to be Gram negative, motile by single polar flagellum, short rods, 1.5–2.3 lm long and 0.8–1.5 lm wide (Fig. 1). The temperature range for growth was found to be 10–40 °C with an optimum of 30 °C. Growth was found to occur at pH 4.0–10.0 (optimum 5.0–9.0) and at 0–9.0 % NaCl (optimum 3.0–6.0 %). The catalase and oxidase activities were found to be positive. Strain JLT2013T was found to hydrolyse aesculin and Tweens 40 and 80. The Voges–Proskauer reaction, nitrate reduction, indole and H2S production were negative. Strain JLT2013T and Parvularcula reference type strains were found to be positive for activity of alkaline and acid phosphatase, leucine arylamidase, naphtol-AS-BI- phosphohydrolase. Furthermore, strain JLT2013T and Parvularcula
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Summed Feature 8
Values are percentages of total fatty acids Strains: 1. JLT2013T; 2. Parvularcula lutaonensis CC-MMS1T; 3. Parvularcula dongshanensis SH25T; 4. Parvularcula bermudensis HTCC2503T All data were obtained from this study for Parvularcula lutaonensis CC-MMS-1T, Parvularcula dongshanensis SH25T and Parvularcula bermudensis HTCC2503T in parallel tests with strain JL2013T under identical conditions - not detected, tr values are less than 1.0 % a
Summed features are groups of two or three fatty acids that cannot be separated by GLC with the MIDI System. Summed feature 8, C18:1x7c and/or C18:1x6c
reference type strains were found to be negative for hydrolysis of casein, starch, and gelatin; nitrate reduction and H2S production; assimilation of Dmannose, capric acid, malic acid, trisodium citrate and phenylacetic acid; acid production from glucose, mannitol, inositol, sorbitol, rhamnose, sucrose and amygdalin; and activity of lipase (C14), N-acetyl-bglucosaminidase, a-mannosidase and b–fucosidase. However, only strain JLT2013T was found to be
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249
84 63 93 92 92 90 100
100 100 65
Mesorhizobium australicum WSM2073T (AY601516) Nitratireductor basaltis J3T (EU143347) Shinella granuli Ch06T (AY995149) Afifella pfennigii DSM17143T (FR733717) Lutibaculum baratangense AMV1T (FN297835) Parvibaculum indicum P31T (FJ182044) Parvibaculum lavamentivorans DS-1T (CP000774) Parvularcula bermudensis HTCC2503T (AF544015) Parvularcula dongshanensis SH25T (JQ778314) Parvularcula oceanus JLT2013T (JX845635) Parvularcula lutaonensis CC-MMS-1T (EU346850) Hellea balneolensis 26III/A02/215T (AY576758) Rickettsia prowazekii ATCC VR-142T (M21789)
0.02
Fig. 2 Neighbour-joining phylogenetic tree showing the relationships between strain JLT2013T and representatives of the class Alphaproteobacteria based on 16S rRNA gene sequences. Only bootstrap values above 50 % are shown (1,000 replications). Rickettsia prowazekii ATCC VR-142T was used as an
outgroup to define the root of the tree. Bar, 0.02 estimated sequence divergence. Filled circles indicate that the corresponding nodes were also recovered in the trees generated with the maximum-likelihood and maximum-parsimony algorithms
positive for acid production from melibiose and activity of valine arylamidase and a-glucosidase. The reference type strains of other Parvularcula species were found to be able to utilize D-glucose whereas strain JLT2013T could not. More detailed results of the physiological characterisations are given in Table 1. Strain JLT2013T showed sensitivity to kanamycin, gentamicin, rifampicin, neomycin and streptomycin. The DNA G?C content of strain JLT2013T was determined to be 66.3 mol%. The polar lipids of strain JLT2013T was identified as diphosphatidylglycerol, phosphatidylglycerol, three sphingoglycolipids and three unknown glycolipids (Fig. S1). The fatty acid profile determined for the novel isolate differed from those of recognized species of the genus Parvularcula (Cho and Giovannoni 2003; Arun et al. 2009; Yu et al. 2013) in the proportions of C16:0, C18:0 and summed feature 8 (C18:1x7c and/or C18:1x6c). Furthermore, C16:0 alcohol, iso-C10:0, anteiso-C14:0, anteiso-C15:0, C14:1x5c and C15:0 3-OH were detected only in cells of strains JLT2013T, whereas C14:0, C17:0 and isoC20:0 were found only in cells of P. dongshanensis SH25T and cyclo-C19:0x8c only in cells of P. bermudensis HTCC2503T (Table 2). Like other species of the genus Parvularcula, the predominant respiratory quinone in strain JLT2013T was found to be Q-10.
Phylogenetic analysis A nearly complete 16S rRNA gene sequence of strain JLT2013T (1426 bp, GenBank accession number: JX845635) was obtained. Phylogenetic distances and clustering obtained via the neighbor-joining (Saitou and Nei 1987) are shown in Fig. 2. As the phylogenetic tree shows, strain JLT2013T fell within a clade comprising species of the genus Parvularcula and formed a coherent cluster with P. lutaonensis CC-MMS-1T. A similar result was observed in the maximum-likelihood and maximum-parsimony tree (Fig. S2, S3), although in these trees strain clustered with P. dongshanensis SH25T. Strain JLT2013T exhibited 96.7 %, 96.0 % and 95.2 % 16S rRNA gene sequence similarity with P. lutaonensis CC-MMS-1T, P. dongshanensis SH25T and P. bermudensis HTCC2503T, respectively. Taxonomic conclusion Based on 16S rRNA gene sequence analysis, strain JLT2013T exhibits high 16S rRNA gene sequence similarity with P. lutaonensis CC-MMS-1T (96.7 %), P. dongshanensis SH25T (96.0 %) and P. bermudensis HTCC2503T (95.2 %). The phylogenetic association of strain JLT2013T with the type strains of members of the genus Parvularcula is consistent as indicated by neighbour-joining, maximum likelihood and
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maximum-parsimony trees (Fig. 1; Fig. S2, S3), although variations in topology are noted. Strain JLT2013T is distinguishable from the members of the genus Parvularcula by differences in polyphasic taxonomic characterization, such as hydrolysis of aesculin and urea, enzymic activity of valine arylamidase, a-glucosidase and melibiose, DNA G?C content and fatty acid composition. Based on phylogenetic analysis and phenotypic characteristics, strain JLT2013T can be considered to represent a novel species of the genus Parvularcula, for which the name P. oceanus sp. nov. is proposed.
phosphatidylglycerol, three sphingoglycolipids and three unknown glycolipids. The predominant cellular fatty acids are C12:0, summed feature 8 as defined by the MIDI system (C18:1x7c and/or C18:1x6c), C10:0, C16:0, and C18:0. The predominant respiratory quinone is Q-10. The DNA G ? C content of the type strain is 66.3 mol%. The type strain is JLT2013T (=LMG 27362T = CGMCC 1.12400T), isolated from deep-sea water in Southeastern Pacific. The GenBank accession number for the 16S rRNA gene sequence of strain JLT2013T is JX845635.
Description of P. oceanus sp. nov.
Acknowledgements This work was supported by the National Key Basic Research Program of China (2013CB955700), the SOA Project (201105021), the National Natural Science Foundation of China project (41276131, 91028001) and MELRI1304.
P. oceanus (o.cean.us. N.L. fem. n. oceanus, the ocean, a bacterium isolated from a marine environment) Cells are Gram-stain negative, aerobic, motile by single polar flagellum, short rods, (1.5–2.3 lm in length and 0.8–1.5 lm in diameter). Colonies incubation on MA are circular, smooth, convex, orangecoloured. Bacterial growth is observed at temperature 10–40 °C (optimum, 30 °C), at pH 4.0–10.0 (optimum, 5.0–9.0) and with 0–9.0 % (w/v) NaCl (optimum, 3.0–6.0 % NaCl). Positive for oxidase and catalase. Poly-b-hydroxybutyrate granules are produced and bacteriochlorophyll a is absent. Aesculin, Tweens 40 and 80 are hydrolysed but gelatin, starch, casein, urea and DNA are not. Nitrate is not reduced. Indole and H2S are not produced. Negative for Voges– Proskauer reaction. According to the API 20E tests, acid is formed from melibiose but not from glucose, mannitol, inositol, sorbitol, rhamnose, sucrose or amygdalin. In assays with the API ZYM system, alcaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, aglucosidase and b-glucosidase are present but lipase (C14), cystine arylamidase, trypsine, a-chymotrypsine, a-galactosidase, b-galactosidase, b-glucuronidase N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase are absent. The following substrates are oxidized as carbon sources in Biolog GN2 tests: Tween-40, Tween-80, adonitol, methyl pyruvate, formic acid, D-galactonic acid lactone, D-gluconicacid, p-hydroxy phenylacetic acid, a-ketovaleric acid, malonic acid, sebacic acid, succinamic acid, glycyl-Lglutamic acid, urocanic acid and glycerol. The polar lipids consist of diphosphatidylglycerol,
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