Antonie van Leeuwenhoek DOI 10.1007/s10482-014-0203-7
ORIGINAL PAPER
Actibacterium atlanticum sp. nov., isolated from surface seawater of the Atlantic Ocean Guizhen Li • Qiliang Lai • Fengqin Sun • Yaping Du • Xiupian Liu • Guangyu Li • Yunbiao Xie • Zongze Shao
Received: 1 February 2014 / Accepted: 23 May 2014 Ó Springer International Publishing Switzerland 2014
Abstract A taxonomic study was carried out on strain 22II-S11-z10T, which was isolated from the surface seawater of the Atlantic Ocean. The bacterium was found to be Gram-stain negative, oxidase and catalase positive, oval- to rod-shaped and non-motile. Growth was observed at salinities of 0.5–9 % and at temperatures of 10–41 °C. The isolate can reduce nitrate to nitrite, degrade gelatin and aesculin, but can not degrade Tween 80. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 22II-S11-z10T belongs to the genus Actibacterium, with the highest sequence similarity to the type strain Actibacterium mucosum CECT 7668T (97.3 %). The DNA–DNA Guizhen Li and Qiliang Lai contributed equally to this work.
hybridization estimate value between strain 22II-S11z10T and A. mucosum CECT 7668T was 19.30 ± 2.29 %. The principal fatty acids were identified as Summed Feature 8 (C18:1x7c/x6c as defined by the MIDI system, 75.2 %) and Summed Feature 3 (C16:1x7c/x6c, 6.9 %). The G?C content of the chromosomal DNA was determined to be 59.0 mol%. The respiratory quinone was determined to be Q-10 (100 %). Phosphatidylglycerol, phosphatidylcholine, two phospholipids, two aminolipids and two lipids were identified in the polar lipids. The combined genotypic and phenotypic data show that strain 22II-S11-z10T represents a novel species within the genus Actibacterium, for which the name Actibacterium atlanticum sp. nov. is proposed, with the type strain 22II-S11-z10T (=MCCC 1A09298T = LMG 27158T).
Transmission electron micrographs of cells of strain 22II-S11z10T, polar lipids of strain 22II-S11-z10T, MaximumLikelihood tree and Minimum Evolution tree are available as supplementary figures in ANTO Online.
Keywords Actibacterium atlanticum Sp. Nov. Atlantic Ocean Taxonomy Surface seawater DDH
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0203-7) contains supplementary material, which is available to authorized users.
Abbreviation MCCC Marine Culture Collection of China
G. Li Q. Lai F. Sun Y. Du X. Liu G. Li Y. Xie Z. Shao (&) State Key Laboratory Breeding Base of Marine Genetic Resources, Key Laboratory of Marine Genetic Resources, Third Institute of Oceanography, SOA, Key Laboratory of Marine Genetic Resources of Fujian Province, Collaborative Innovation Center of Deep Sea Biology, Xiamen 361005, China e-mail: [email protected]
Introduction During an investigation of bacterial diversity in the water column of the Atlantic Ocean, strain 22II-S11z10T was isolated and characterized taxonomically along with many other bacterial isolates. Comparative
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16S rRNA gene sequence analysis indicated that strain 22II-S11-z10T belongs to the genus Actibacterium. The genus Actibacterium was proposed by Lucena and colleagues, and comprises only one species, Actibacterium mucosum (Lucena et al. 2012).The aim of the present work was to determine the exact taxonomic position of strain 22II-S11-z10T by using a polyphasic approach.
Materials and methods Bacterial strains, isolation and cultivation Surface seawater was sampled at the site of 22IIMAR-S11 (W14.4°, S13.9°) in the South Atlantic Ocean on the R/V ‘‘Da-Yang Yi-Hao’’ in Feb, 2011 during cruise COMRA 22II. The surface seawater was diluted and spread on marine agar 2216 medium (BD Difco). After 1 month of aerobic incubation at 25 °C, the colonies were picked. Purity was confirmed by the uniformity of cell morphology after repeated restreaking. For morphological and biochemical characterization, strain 22II-S11-z10T was cultivated on marine agar 2216 medium (BD Difco) unless otherwise indicated. A. mucosum CECT 7668T was used as the reference strain under comparable culture conditions. Phenotypic characterization Gram staining was performed using a Gram staining kit (Hangzhou Tianhe Microorganism Reagent Co.) according to the manufacturer’s instructions. Catalase activity was determined by bubble production in 10 % (v/v) hydrogen peroxide solution. Oxidase activity was evaluated by the oxidation of 1 % (w/v) N,N,N’,N’tetramethyl-1,4-phenylenediamine. The cell size, morphology and flagellation pattern were determined by transmission electron microscopy (JEM-1230, JEOL) after cells had been negatively stained with phosphotungstic acid. Cultures were grown on marine agar at 28 °C for 1 day prior to staining. Cell motility was observed by the hanging-drop method (Skerman 1967). The temperature range for growth was determined in marine broth 2216 by incubating cultures at 4–55 °C. Growth with NaCl as the sole salt was investigated using Luria–Bertani medium (Sambrook et al. 1989) supplemented with a NaCl concentrations of 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18 or 20 %
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(w/v). The pH range for growth was determined in marine broth 2216 at pH 2.0–10.0 at intervals of 1 pH unit with citrate/phosphate (pH 2.0–7.0), Tris/HCl (pH 8.0–9.0), or sodium carbonate/sodium bicarbonate (pH 10.0) buffers. Antibiotic susceptibility tests were performed using the disc diffusion method as described previously (Shieh et al. 2003). Hydrolysis of DNA, starch, dried skimmed milk, cellulose and Tween 20, 40, 60 and 80 were tested on marine agar 2216 plates supplemented with 0.2 % DNA, 0.2 % (w/v) soluble starch, 0.5 % dried skimmed milk, 0.8 % cellulose or 0.5 % (v/v) Tween 20, 40, 60 and 80, according to standard methods (Dong and Cai 2001). API 20NE, API 20E, and API ZYM strips (bioMe´rieux) were carried out according to the manufacturer’s instructions, with the single modification of adjusting the NaCl concentration to 3.0 % in all tests. Determination of 16S rRNA gene sequence and phylogenetic analysis Genomic DNA was extracted according to a previously described method (Ausubel et al. 1995) and the 16S rRNA gene was amplified by PCR using primers described previously (Liu and Shao 2005). Sequence similarity was determined using the EzTaxon-e server (Kim et al. 2012). Phylogenetic analysis was performed using MEGA version 5.0 (Tamura et al. 2011) with distance options according to the Kimura twoparameter model and clustering with the neighbourjoining (Saitou and Nei 1987), minimum evolution (Rzhetsky and Nei 1992) and maximum likelihood (Felsenstein 1981) methods, and supported with bootstrap values which were calculated based on 1,000 replications. Sequences of related taxa were obtained from the GenBank database. Genome sequencing, G?C content, DDH and ANI estimate The genome sequences strain 22II-S11-z10T were determined by Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China), using Solexa paired-end (500 bp library) sequencing technology (Illumina, SanDiego, CA, USA). The G?C content of the chromosomal DNA was determined by analysis of the draft genome sequence. DNA–DNA hybridization (DDH) estimate value was analyzed using the genome-to-genome distance
Antonie van Leeuwenhoek
calculator (GGDC2.0) with the alignment method of BLAST? (Auch et al. 2010a, b; Meier-Kolthoff et al. 2013). The average nucleotide identity (ANI) was calculated using the algorithm of Goris et al. (2007) by the web service of EZGenome.
Table 1 Differential characteristics between strain 22II-S11z10T and Actibacterium mucosum CECT 7668T
Determination of fatty acid, isoprenoid ubiquinone, and polar lipid compositions Fatty acids in whole cells grown on marine agar 2216 medium at 28 °C for 48 h were saponified, methylated, and extracted using the standard protocol of MIDI (Sherlock Microbial Identification System, version 6.0B). The fatty acids were analysed by gas chromatography (Agilent Technologies 6850) and identified using the TSBA6.0 database of the MIDI System (Sasser 1990). Analysis of the respiratory quinone and polar lipids were carried out by the Identification Service of the DSMZ, Braunschweig, Germany.
Results and discussion
1
2
Cell
0.7–0.8a
0.2–0.5a
1.3–1.8
0.6–1.4
Temperature range (°C)
10–41
15–37
NaCl range for growth (%)
0.5–9 (1–7)
1.7–5
API ZYM w
–
Cystine aminopeptidase, trypsin, agalactosidase
–
w
a-Glucosidase
w
?
Reduction of nitrate
?
–
Malic acid
?
–
–
?
?
–
Phosphatidylglycerol
?
?
Phosphatidylcholine Aminolipids
? ?
– ?
Phospholipids
?
?
Glycolipid
–
?
Unknown lipid
?
?
DNA G?C content (mol%)
59.0
61.3 (60.8a)
API 20NE
API 20E Fermentation of melibiose Susceptibility Gentamicin (10), kanamycin (30), cotrimoxazole (25) Lipid
Phenotypic characteristics Strain 22II-S11-z10T was found to be Gram-negative, non-spore-forming, rod-shaped, and non-motile, 0.7–0.8 lm in width and 1.3–1.8 lm in length (see Supplementary Materials Fig. S1). On marine agar 2216, strain 22II-S11-z10T forms smooth grey-white colonies with convex and regular edges of 1 mm in diameter after 2 days incubation at 28 °C. Moderately halophilic, 22IIS11-z10T can grow in media with 0.5–9.0 % NaCl (optimum 1–7 %) at 10–41 °C (optimum 28 °C), at a pH of 4.0–9.0 (optimum 7.0–8.0). It cannot hydrolyze Tween 20, 40, 60 and 80, starch, dried skimmed milk, DNA and cellulose. Strain 22II-S11-z10T is sensitive to ampicillin (10 lg/per disk, OXOID), chloromycetin (30), carbenicillin (100), cephradin (30), cefobid (30), ciprofloxacin (5), cefalexin (30), gentamicin (10), rocephin (30), vibramycin (30), erythromycin (15), kanamycin (30), cefazolin (30), minomycin (30), norfloxacin (10), ofloxacin (5), oxacillin (1), penicillin G (10), polymyxin B (30 IU), piperacillin (100), rifampicin (5), streptomycin (10), tetracycline (30), co-trimoxazole (25) and vancomycin (30); resistant to clindamycin (2), metronidazole (5) and lincomycin(2). Other characteristics are given in the species description and Table 1.
Lipase (C14)
a
Data from draft genome sequence
Strains: 1, 22II-S11-z10T; 2, A. mucosum CECT 7668T (Lucena et al. 2012) The data of API 20NE, API 20E, API ZYM and antibiotic susceptibility tests were examined in this study. Characteristics are scored as: ?, positive; -, negative; w, weak
16S rRNA gene sequence analysis A nearly full-length 16S rRNA gene sequence (1,430 nt, GenBank accession number KJ159064.) from strain 22II-S11-z10T was determined. The 22IIS11-z10T 16S rRNA gene showed the highest sequence similarity to that of A. mucosum CECT 7668T (97.3 %), followed by Shimia isoporae SW6T (96.1 %). Sequence similarity to the type strains of other species were all below 96.0 %. As shown in Fig. 1, a phylogenetic tree was constructed based on
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Antonie van Leeuwenhoek Fig. 1 Neighbour-joining tree showing the phylogenetic positions of strain 22II-S11-z10T and the species of the genus Actibacterium and related genera of family Rhodobacteraceae, based on 16S rRNA gene sequences. Filled circles indicate nodes that were also recovered in maximumlikelihood and minimum evolution trees based on the same sequences. Bootstrap values (expressed as percentages of 1,000 replications) are shown at branch points. Bar, 0.01 nucleotide substitution rate (Knuc) units. Oceanibaculum indicum P24T (EU656113) was used as outgroup
the 16S rRNA gene sequences of the genus Actibacterium and related genera of family Rhodobacteraceae. The analysis showed that strain 22II-S11-z10T forms a clade with A. mucosum CECT 7668T. This topology was confirmed by the maximum-likelihood and minimum evolution trees (Fig. 1; Supplementary Materials Figs. S2 and S3).
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Genome sequencing, DNA G?C content, DDH and ANI estimate A total of 500 Mbp clean data for strain 22II-S11-z10T was generated to reach about 100-fold depth of coverage with an Illumina/Solexa Genome Analyzer IIx. The clean data was assembled by SOAPdenovo2 (Luo et al.
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2012). The accession number for strain 22II-S11-z10T is AQQY00000000. The draft genome sequence of A. mucosum CECT 7668T (JFKE00000000) was obtained from NCBI. The chromosomal DNA G?C content of strain 22II-S11-z10T was determined to be 59.0 mol%, which is close to the values determined for A. mucosum CECT 7668T (60.8 mol% according to draft genome sequence; 61.3 mol% according to Lucena et al. (2012)). The DDH estimated value between strain 22II-S11-z10T and A. mucosum CECT 7668T was 19.30 ± 2.29 %, which is below the standard cut-off value (70 %) (Wayne et al. 1987). The ANI values between strain 22II-S11-z10T and A. mucosum CECT 7668T were 71.74–71.79 %, which is below standard ANI criteria for species identity (95–96 %) (Richter and Rossello-Mora 2009). These data confirm that strain 22II-S11-z10T represents a novel species of the genus Actibacterium. Chemotaxonomic characteristics The principal fatty acids of strain 22II-S11-z10T were identified as Summed Feature 8 (C18:1x7c/x6c, as defined by MIDI; 75.2 %) and Summed Feature 3 (C16:1x7c/x6c, 6.9 %). In addition, the major fatty acids of A. mucosum CECT 7668T were identified as Summed Feature 8 (C18:1x7c/x6c, 65.5 %), C16:0 (12.0 %), C10:0 3-OH (7.2 %) and C12:0 3-OH (5.3 %). The major fatty acid profile of strain 22II-S11-z10T is in good agreement with that of the type strain of the type species of the genus Actibacterium but it can be differentiated from the type strain by the percentage of Summed Feature 8 (C18:1x7c/x6c), Summed Feature 3 (C16:1x7c/x6c) and C12:0 3-OH (Table 2). The respiratory quinone of strain 22II-S11-z10T was determined to be Q-10 (100 %). This is consistent with the genus Actibacterium (Lucena et al. 2012). Strain 22II-S11-z10T was found to contain phosphatidylglycerol (PG), phosphatidylcholine (PC), two phospholipids, two unknown aminolipids and two unknown lipids (shown in Supplementary Materials Fig. S4). Strain 22II-S11-z10T can be differentiated from A. mucosum CECT 7668T by the presence of PC and the absence of glycolipid. Taxonomic conclusion The results of the phylogenetic analysis, phenotypic analysis, and chemotaxonomic studies presented
Table 2 Fatty acids of strain 22II-S11-z10T and Actibacterium mucosum CECT 7668T Fatty acid
1
2
C10:0 3-OH
2.4
7.2
C12:0 3-OH
ND
5.3
C16:0
2.9
12.0
C17:1x7c
ND
1.6
C18:0
4.2
2.4
C18:1x7c 11-methyl
1.3
1.5 tr
C19:0 cyclo x8c
2.6
Sum in Feature 2 (C14:0 3OH/C16:1 iso I)
1.6
ND
Sum in Feature 3 (C16:1x7c/x6c)
6.9
tr
Sum in Feature 8 (C18:1x7c/x6c)
75.2
T
65.5 T
Strains: 1, 22II-S11-z10 ; 2, A. mucosum CECT 7668 . The data were obtained in this study. Values are percentages of total fatty acids. ND not detected, tr trace amount (\1.0 %)
above support the view that strain 22II-S11-z10T should be assigned to the genus Actibacterium. However, the strain could be distinguished from the closelyrelated species A. mucosum CECT 7668T by some phenotypic characteristics given in Table 1. Considering its low DDH and ANI values when compared with the closest related species, the isolate cannot be assigned to any previously recognized species. Therefore, strain 22II-S11-z10T represents a novel species of the genus Actibacterium, for which the name Actibacterium atlanticum sp. nov. is proposed. Description of Actibacterium atlanticum sp. nov Actibacterium atlanticum (at.lan’ti.cum. L. neutr. adj. atlanticum Atlantic, from the Atlantic Ocean). Cells are Gram-negative, oval- to rod-shaped, 0.7–0.8 lm in width and 1.3–1.8 lm in length. Nonmotile. Positive for oxidase, catalase, acetoin production (Voges Proskauer), gelatinase, b-glucosidase (aesculin hydrolysis), b-galactosidase and reduction of nitrate to nitrite, but negative for D-glucose fermentation, rhamnose fermentation, melibiose fermentation, amygdalin fermentation, arabinose fermentation, arginine dihydrolase, urease, ornithine decarboxylase, citrate utilization, denitrification, indole production, lysine decarboxylase, H2S production, tryptophan deaminase, mannitol fermentation, inositol fermentation, sorbitol fermentation and saccharose fermentation. Cannot hydrolyze DNA, Tween 20, 40, 60 and 80, starch, dried skimmed milk or cellulose. On marine agar 2216, forms
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smooth grey colonies with regular edges, slightly raised in the centre, of 0.5–1 mm in diameter after 48 h incubation at 28 °C. Moderately halophilic, grows in 0.5–9.0 % NaCl (optimum 1–7 %), at 10–41 °C (optimum 28 °C) and at a pH range of 4.0–9.0 (optimum 7.0–8.0). Principal fatty acids are Summed Feature 8 (C18:1x7c/x6c as defined by the MIDI system) and Summed Feature 3 (C16:1x7c/x6c). The respiratory quinone is Q-10 (100 %). The polar lipids are phosphatidylglycerol, phosphatidylcholine, two phospholipids, two aminolipids and two lipids. API ZYM test strip results indicate that the type strain is positive for alkaline phosphatase, acid phosphatase, esterase (C4), esterase lipase (C8), leucine aminopeptidase, valine aminopeptidase; weakly positive for lipase (C14), a-glucosidase; negative for cystine aminopeptidase, a-chymotrypsin, a-fucosidase, trypsin, naphtol-AS-Bl-phosphoamidase, N-acetyl-b-glucosaminidase, a-galactosidase, a-mannosidase, b-galactosidase, b-glucosidase or b-glucuronidase. The API 20NE showed that the type strain can utilize malic acid, but cannot utilize D-mannitol, potassium gluconate, N-acetyl-glucosamine, adipic acid, capric acid, D-glucose, D-maltose, D-mannose, L-arabinose, phenylacetic acid or trisodium citrate. The G?C content of the chromosomal DNA of the type strain is 59.0 mol%. Table 1 shows the characteristics used to distinguish the type strain from related species. The type strain 22II-S11-z10T (=MCCC 1A09298T = LMG 27158T) was isolated from surface seawater of the Atlantic Ocean. The GenBank accession number for the 16S rRNA gene sequence of A. atlanticum 22II-S11-z10T is KJ159064. Acknowledgments This work was financially supported by COMRA program (No. DY125-15-R-01), Public Welfare Project of SOA (201005032) and National Infrastructure of Natural Resources for Science and Technology Program of China (No. NIMR-2013-9).
References Auch AF, Klenk HP, Goker M (2010a) Standard operating procedure for calculating genome-to-genome distances based on high-scoring segment pairs. Stand Genomic Sci 2:142–148 Auch AF, von Jan M, Klenk HP, Goker M (2010b) Digital DNA–DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand Genomic Sci 2:117–134 Ausubel F, Brent R, Kingston R, Moore D, Seidman J, Smith J, Struhl K (1995) Short protocols in molecular biology: a compendium of methods from Current protocols in molecular biology, 3rd edn. Wiley, New York
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Dong X-Z, Cai M-Y (2001) Determinative manual for routine bacteriology. Scientific Press, Beijing (English translation) Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17:368–376 Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM (2007) DNA–DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol 57:81–91 Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, Won S, Chun J (2012) Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62:716–721 Liu C, Shao Z (2005) Alcanivorax dieselolei sp. nov., a novel alkane-degrading bacterium isolated from sea water and deep-sea sediment. Int J Syst Evol Microbiol 55:1181–1186 Lucena T, Ruvira MA, Garay E, Macian MC, Arahal DR, Pujalte MJ (2012) Actibacterium mucosum gen. nov., sp. nov., a marine alphaproteobacterium from Mediterranean seawater. Int J Syst Evol Microbiol 62:2858–2864 Luo R, Liu B, Xie Y, Li Z, Huang W, Yuan J, He G, Chen Y, Pan Q, Liu Y, Tang J, Wu G, Zhang H, Shi Y, Liu Y, Yu C, Wang B, Lu Y, Han C, Cheung DW, Yiu SM, Peng S, Xiaoqian Z, Liu G, Liao X, Li Y, Yang H, Wang J, Lam TW, Wang J (2012) SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. Gigascience 1:18 Meier-Kolthoff JP, Auch AF, Klenk HP, Goker M (2013) Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinform 14:60 Richter M, Rossello-Mora R (2009) Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA 106:19126–19131 Rzhetsky A, Nei M (1992) Statistical properties of the ordinary least-squares, generalized least-squares, and minimumevolution methods of phylogenetic inference. J Mol Evol 35:367–375 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids, MIDI Technical Note 101. MIDI, Newark Shieh WY, Chen YW, Chaw SM, Chiu HH (2003) Vibrio ruber sp. nov., a red, facultatively anaerobic, marine bacterium isolated from sea water. Int J Syst Evol Microbiol 53:479–484 Skerman VBD (1967) A guide to the identification of the genera of bacteria, 2nd edn. Williams & Wilkins, Baltimore Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 Wayne LG, Brenner DJ, Colwell RR, Grimont PAD, Kandler O, Krichevsky MI, Moore LH, Moore WEC, Murray RGE, Stackebrandt E, Starr MP, Tru¨per HG (1987) Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37:463–464
ORIGINAL PAPER
Actibacterium atlanticum sp. nov., isolated from surface seawater of the Atlantic Ocean Guizhen Li • Qiliang Lai • Fengqin Sun • Yaping Du • Xiupian Liu • Guangyu Li • Yunbiao Xie • Zongze Shao
Received: 1 February 2014 / Accepted: 23 May 2014 Ó Springer International Publishing Switzerland 2014
Abstract A taxonomic study was carried out on strain 22II-S11-z10T, which was isolated from the surface seawater of the Atlantic Ocean. The bacterium was found to be Gram-stain negative, oxidase and catalase positive, oval- to rod-shaped and non-motile. Growth was observed at salinities of 0.5–9 % and at temperatures of 10–41 °C. The isolate can reduce nitrate to nitrite, degrade gelatin and aesculin, but can not degrade Tween 80. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 22II-S11-z10T belongs to the genus Actibacterium, with the highest sequence similarity to the type strain Actibacterium mucosum CECT 7668T (97.3 %). The DNA–DNA Guizhen Li and Qiliang Lai contributed equally to this work.
hybridization estimate value between strain 22II-S11z10T and A. mucosum CECT 7668T was 19.30 ± 2.29 %. The principal fatty acids were identified as Summed Feature 8 (C18:1x7c/x6c as defined by the MIDI system, 75.2 %) and Summed Feature 3 (C16:1x7c/x6c, 6.9 %). The G?C content of the chromosomal DNA was determined to be 59.0 mol%. The respiratory quinone was determined to be Q-10 (100 %). Phosphatidylglycerol, phosphatidylcholine, two phospholipids, two aminolipids and two lipids were identified in the polar lipids. The combined genotypic and phenotypic data show that strain 22II-S11-z10T represents a novel species within the genus Actibacterium, for which the name Actibacterium atlanticum sp. nov. is proposed, with the type strain 22II-S11-z10T (=MCCC 1A09298T = LMG 27158T).
Transmission electron micrographs of cells of strain 22II-S11z10T, polar lipids of strain 22II-S11-z10T, MaximumLikelihood tree and Minimum Evolution tree are available as supplementary figures in ANTO Online.
Keywords Actibacterium atlanticum Sp. Nov. Atlantic Ocean Taxonomy Surface seawater DDH
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0203-7) contains supplementary material, which is available to authorized users.
Abbreviation MCCC Marine Culture Collection of China
G. Li Q. Lai F. Sun Y. Du X. Liu G. Li Y. Xie Z. Shao (&) State Key Laboratory Breeding Base of Marine Genetic Resources, Key Laboratory of Marine Genetic Resources, Third Institute of Oceanography, SOA, Key Laboratory of Marine Genetic Resources of Fujian Province, Collaborative Innovation Center of Deep Sea Biology, Xiamen 361005, China e-mail: [email protected]
Introduction During an investigation of bacterial diversity in the water column of the Atlantic Ocean, strain 22II-S11z10T was isolated and characterized taxonomically along with many other bacterial isolates. Comparative
123
Antonie van Leeuwenhoek
16S rRNA gene sequence analysis indicated that strain 22II-S11-z10T belongs to the genus Actibacterium. The genus Actibacterium was proposed by Lucena and colleagues, and comprises only one species, Actibacterium mucosum (Lucena et al. 2012).The aim of the present work was to determine the exact taxonomic position of strain 22II-S11-z10T by using a polyphasic approach.
Materials and methods Bacterial strains, isolation and cultivation Surface seawater was sampled at the site of 22IIMAR-S11 (W14.4°, S13.9°) in the South Atlantic Ocean on the R/V ‘‘Da-Yang Yi-Hao’’ in Feb, 2011 during cruise COMRA 22II. The surface seawater was diluted and spread on marine agar 2216 medium (BD Difco). After 1 month of aerobic incubation at 25 °C, the colonies were picked. Purity was confirmed by the uniformity of cell morphology after repeated restreaking. For morphological and biochemical characterization, strain 22II-S11-z10T was cultivated on marine agar 2216 medium (BD Difco) unless otherwise indicated. A. mucosum CECT 7668T was used as the reference strain under comparable culture conditions. Phenotypic characterization Gram staining was performed using a Gram staining kit (Hangzhou Tianhe Microorganism Reagent Co.) according to the manufacturer’s instructions. Catalase activity was determined by bubble production in 10 % (v/v) hydrogen peroxide solution. Oxidase activity was evaluated by the oxidation of 1 % (w/v) N,N,N’,N’tetramethyl-1,4-phenylenediamine. The cell size, morphology and flagellation pattern were determined by transmission electron microscopy (JEM-1230, JEOL) after cells had been negatively stained with phosphotungstic acid. Cultures were grown on marine agar at 28 °C for 1 day prior to staining. Cell motility was observed by the hanging-drop method (Skerman 1967). The temperature range for growth was determined in marine broth 2216 by incubating cultures at 4–55 °C. Growth with NaCl as the sole salt was investigated using Luria–Bertani medium (Sambrook et al. 1989) supplemented with a NaCl concentrations of 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18 or 20 %
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(w/v). The pH range for growth was determined in marine broth 2216 at pH 2.0–10.0 at intervals of 1 pH unit with citrate/phosphate (pH 2.0–7.0), Tris/HCl (pH 8.0–9.0), or sodium carbonate/sodium bicarbonate (pH 10.0) buffers. Antibiotic susceptibility tests were performed using the disc diffusion method as described previously (Shieh et al. 2003). Hydrolysis of DNA, starch, dried skimmed milk, cellulose and Tween 20, 40, 60 and 80 were tested on marine agar 2216 plates supplemented with 0.2 % DNA, 0.2 % (w/v) soluble starch, 0.5 % dried skimmed milk, 0.8 % cellulose or 0.5 % (v/v) Tween 20, 40, 60 and 80, according to standard methods (Dong and Cai 2001). API 20NE, API 20E, and API ZYM strips (bioMe´rieux) were carried out according to the manufacturer’s instructions, with the single modification of adjusting the NaCl concentration to 3.0 % in all tests. Determination of 16S rRNA gene sequence and phylogenetic analysis Genomic DNA was extracted according to a previously described method (Ausubel et al. 1995) and the 16S rRNA gene was amplified by PCR using primers described previously (Liu and Shao 2005). Sequence similarity was determined using the EzTaxon-e server (Kim et al. 2012). Phylogenetic analysis was performed using MEGA version 5.0 (Tamura et al. 2011) with distance options according to the Kimura twoparameter model and clustering with the neighbourjoining (Saitou and Nei 1987), minimum evolution (Rzhetsky and Nei 1992) and maximum likelihood (Felsenstein 1981) methods, and supported with bootstrap values which were calculated based on 1,000 replications. Sequences of related taxa were obtained from the GenBank database. Genome sequencing, G?C content, DDH and ANI estimate The genome sequences strain 22II-S11-z10T were determined by Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China), using Solexa paired-end (500 bp library) sequencing technology (Illumina, SanDiego, CA, USA). The G?C content of the chromosomal DNA was determined by analysis of the draft genome sequence. DNA–DNA hybridization (DDH) estimate value was analyzed using the genome-to-genome distance
Antonie van Leeuwenhoek
calculator (GGDC2.0) with the alignment method of BLAST? (Auch et al. 2010a, b; Meier-Kolthoff et al. 2013). The average nucleotide identity (ANI) was calculated using the algorithm of Goris et al. (2007) by the web service of EZGenome.
Table 1 Differential characteristics between strain 22II-S11z10T and Actibacterium mucosum CECT 7668T
Determination of fatty acid, isoprenoid ubiquinone, and polar lipid compositions Fatty acids in whole cells grown on marine agar 2216 medium at 28 °C for 48 h were saponified, methylated, and extracted using the standard protocol of MIDI (Sherlock Microbial Identification System, version 6.0B). The fatty acids were analysed by gas chromatography (Agilent Technologies 6850) and identified using the TSBA6.0 database of the MIDI System (Sasser 1990). Analysis of the respiratory quinone and polar lipids were carried out by the Identification Service of the DSMZ, Braunschweig, Germany.
Results and discussion
1
2
Cell
0.7–0.8a
0.2–0.5a
1.3–1.8
0.6–1.4
Temperature range (°C)
10–41
15–37
NaCl range for growth (%)
0.5–9 (1–7)
1.7–5
API ZYM w
–
Cystine aminopeptidase, trypsin, agalactosidase
–
w
a-Glucosidase
w
?
Reduction of nitrate
?
–
Malic acid
?
–
–
?
?
–
Phosphatidylglycerol
?
?
Phosphatidylcholine Aminolipids
? ?
– ?
Phospholipids
?
?
Glycolipid
–
?
Unknown lipid
?
?
DNA G?C content (mol%)
59.0
61.3 (60.8a)
API 20NE
API 20E Fermentation of melibiose Susceptibility Gentamicin (10), kanamycin (30), cotrimoxazole (25) Lipid
Phenotypic characteristics Strain 22II-S11-z10T was found to be Gram-negative, non-spore-forming, rod-shaped, and non-motile, 0.7–0.8 lm in width and 1.3–1.8 lm in length (see Supplementary Materials Fig. S1). On marine agar 2216, strain 22II-S11-z10T forms smooth grey-white colonies with convex and regular edges of 1 mm in diameter after 2 days incubation at 28 °C. Moderately halophilic, 22IIS11-z10T can grow in media with 0.5–9.0 % NaCl (optimum 1–7 %) at 10–41 °C (optimum 28 °C), at a pH of 4.0–9.0 (optimum 7.0–8.0). It cannot hydrolyze Tween 20, 40, 60 and 80, starch, dried skimmed milk, DNA and cellulose. Strain 22II-S11-z10T is sensitive to ampicillin (10 lg/per disk, OXOID), chloromycetin (30), carbenicillin (100), cephradin (30), cefobid (30), ciprofloxacin (5), cefalexin (30), gentamicin (10), rocephin (30), vibramycin (30), erythromycin (15), kanamycin (30), cefazolin (30), minomycin (30), norfloxacin (10), ofloxacin (5), oxacillin (1), penicillin G (10), polymyxin B (30 IU), piperacillin (100), rifampicin (5), streptomycin (10), tetracycline (30), co-trimoxazole (25) and vancomycin (30); resistant to clindamycin (2), metronidazole (5) and lincomycin(2). Other characteristics are given in the species description and Table 1.
Lipase (C14)
a
Data from draft genome sequence
Strains: 1, 22II-S11-z10T; 2, A. mucosum CECT 7668T (Lucena et al. 2012) The data of API 20NE, API 20E, API ZYM and antibiotic susceptibility tests were examined in this study. Characteristics are scored as: ?, positive; -, negative; w, weak
16S rRNA gene sequence analysis A nearly full-length 16S rRNA gene sequence (1,430 nt, GenBank accession number KJ159064.) from strain 22II-S11-z10T was determined. The 22IIS11-z10T 16S rRNA gene showed the highest sequence similarity to that of A. mucosum CECT 7668T (97.3 %), followed by Shimia isoporae SW6T (96.1 %). Sequence similarity to the type strains of other species were all below 96.0 %. As shown in Fig. 1, a phylogenetic tree was constructed based on
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Antonie van Leeuwenhoek Fig. 1 Neighbour-joining tree showing the phylogenetic positions of strain 22II-S11-z10T and the species of the genus Actibacterium and related genera of family Rhodobacteraceae, based on 16S rRNA gene sequences. Filled circles indicate nodes that were also recovered in maximumlikelihood and minimum evolution trees based on the same sequences. Bootstrap values (expressed as percentages of 1,000 replications) are shown at branch points. Bar, 0.01 nucleotide substitution rate (Knuc) units. Oceanibaculum indicum P24T (EU656113) was used as outgroup
the 16S rRNA gene sequences of the genus Actibacterium and related genera of family Rhodobacteraceae. The analysis showed that strain 22II-S11-z10T forms a clade with A. mucosum CECT 7668T. This topology was confirmed by the maximum-likelihood and minimum evolution trees (Fig. 1; Supplementary Materials Figs. S2 and S3).
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Genome sequencing, DNA G?C content, DDH and ANI estimate A total of 500 Mbp clean data for strain 22II-S11-z10T was generated to reach about 100-fold depth of coverage with an Illumina/Solexa Genome Analyzer IIx. The clean data was assembled by SOAPdenovo2 (Luo et al.
Antonie van Leeuwenhoek
2012). The accession number for strain 22II-S11-z10T is AQQY00000000. The draft genome sequence of A. mucosum CECT 7668T (JFKE00000000) was obtained from NCBI. The chromosomal DNA G?C content of strain 22II-S11-z10T was determined to be 59.0 mol%, which is close to the values determined for A. mucosum CECT 7668T (60.8 mol% according to draft genome sequence; 61.3 mol% according to Lucena et al. (2012)). The DDH estimated value between strain 22II-S11-z10T and A. mucosum CECT 7668T was 19.30 ± 2.29 %, which is below the standard cut-off value (70 %) (Wayne et al. 1987). The ANI values between strain 22II-S11-z10T and A. mucosum CECT 7668T were 71.74–71.79 %, which is below standard ANI criteria for species identity (95–96 %) (Richter and Rossello-Mora 2009). These data confirm that strain 22II-S11-z10T represents a novel species of the genus Actibacterium. Chemotaxonomic characteristics The principal fatty acids of strain 22II-S11-z10T were identified as Summed Feature 8 (C18:1x7c/x6c, as defined by MIDI; 75.2 %) and Summed Feature 3 (C16:1x7c/x6c, 6.9 %). In addition, the major fatty acids of A. mucosum CECT 7668T were identified as Summed Feature 8 (C18:1x7c/x6c, 65.5 %), C16:0 (12.0 %), C10:0 3-OH (7.2 %) and C12:0 3-OH (5.3 %). The major fatty acid profile of strain 22II-S11-z10T is in good agreement with that of the type strain of the type species of the genus Actibacterium but it can be differentiated from the type strain by the percentage of Summed Feature 8 (C18:1x7c/x6c), Summed Feature 3 (C16:1x7c/x6c) and C12:0 3-OH (Table 2). The respiratory quinone of strain 22II-S11-z10T was determined to be Q-10 (100 %). This is consistent with the genus Actibacterium (Lucena et al. 2012). Strain 22II-S11-z10T was found to contain phosphatidylglycerol (PG), phosphatidylcholine (PC), two phospholipids, two unknown aminolipids and two unknown lipids (shown in Supplementary Materials Fig. S4). Strain 22II-S11-z10T can be differentiated from A. mucosum CECT 7668T by the presence of PC and the absence of glycolipid. Taxonomic conclusion The results of the phylogenetic analysis, phenotypic analysis, and chemotaxonomic studies presented
Table 2 Fatty acids of strain 22II-S11-z10T and Actibacterium mucosum CECT 7668T Fatty acid
1
2
C10:0 3-OH
2.4
7.2
C12:0 3-OH
ND
5.3
C16:0
2.9
12.0
C17:1x7c
ND
1.6
C18:0
4.2
2.4
C18:1x7c 11-methyl
1.3
1.5 tr
C19:0 cyclo x8c
2.6
Sum in Feature 2 (C14:0 3OH/C16:1 iso I)
1.6
ND
Sum in Feature 3 (C16:1x7c/x6c)
6.9
tr
Sum in Feature 8 (C18:1x7c/x6c)
75.2
T
65.5 T
Strains: 1, 22II-S11-z10 ; 2, A. mucosum CECT 7668 . The data were obtained in this study. Values are percentages of total fatty acids. ND not detected, tr trace amount (\1.0 %)
above support the view that strain 22II-S11-z10T should be assigned to the genus Actibacterium. However, the strain could be distinguished from the closelyrelated species A. mucosum CECT 7668T by some phenotypic characteristics given in Table 1. Considering its low DDH and ANI values when compared with the closest related species, the isolate cannot be assigned to any previously recognized species. Therefore, strain 22II-S11-z10T represents a novel species of the genus Actibacterium, for which the name Actibacterium atlanticum sp. nov. is proposed. Description of Actibacterium atlanticum sp. nov Actibacterium atlanticum (at.lan’ti.cum. L. neutr. adj. atlanticum Atlantic, from the Atlantic Ocean). Cells are Gram-negative, oval- to rod-shaped, 0.7–0.8 lm in width and 1.3–1.8 lm in length. Nonmotile. Positive for oxidase, catalase, acetoin production (Voges Proskauer), gelatinase, b-glucosidase (aesculin hydrolysis), b-galactosidase and reduction of nitrate to nitrite, but negative for D-glucose fermentation, rhamnose fermentation, melibiose fermentation, amygdalin fermentation, arabinose fermentation, arginine dihydrolase, urease, ornithine decarboxylase, citrate utilization, denitrification, indole production, lysine decarboxylase, H2S production, tryptophan deaminase, mannitol fermentation, inositol fermentation, sorbitol fermentation and saccharose fermentation. Cannot hydrolyze DNA, Tween 20, 40, 60 and 80, starch, dried skimmed milk or cellulose. On marine agar 2216, forms
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Antonie van Leeuwenhoek
smooth grey colonies with regular edges, slightly raised in the centre, of 0.5–1 mm in diameter after 48 h incubation at 28 °C. Moderately halophilic, grows in 0.5–9.0 % NaCl (optimum 1–7 %), at 10–41 °C (optimum 28 °C) and at a pH range of 4.0–9.0 (optimum 7.0–8.0). Principal fatty acids are Summed Feature 8 (C18:1x7c/x6c as defined by the MIDI system) and Summed Feature 3 (C16:1x7c/x6c). The respiratory quinone is Q-10 (100 %). The polar lipids are phosphatidylglycerol, phosphatidylcholine, two phospholipids, two aminolipids and two lipids. API ZYM test strip results indicate that the type strain is positive for alkaline phosphatase, acid phosphatase, esterase (C4), esterase lipase (C8), leucine aminopeptidase, valine aminopeptidase; weakly positive for lipase (C14), a-glucosidase; negative for cystine aminopeptidase, a-chymotrypsin, a-fucosidase, trypsin, naphtol-AS-Bl-phosphoamidase, N-acetyl-b-glucosaminidase, a-galactosidase, a-mannosidase, b-galactosidase, b-glucosidase or b-glucuronidase. The API 20NE showed that the type strain can utilize malic acid, but cannot utilize D-mannitol, potassium gluconate, N-acetyl-glucosamine, adipic acid, capric acid, D-glucose, D-maltose, D-mannose, L-arabinose, phenylacetic acid or trisodium citrate. The G?C content of the chromosomal DNA of the type strain is 59.0 mol%. Table 1 shows the characteristics used to distinguish the type strain from related species. The type strain 22II-S11-z10T (=MCCC 1A09298T = LMG 27158T) was isolated from surface seawater of the Atlantic Ocean. The GenBank accession number for the 16S rRNA gene sequence of A. atlanticum 22II-S11-z10T is KJ159064. Acknowledgments This work was financially supported by COMRA program (No. DY125-15-R-01), Public Welfare Project of SOA (201005032) and National Infrastructure of Natural Resources for Science and Technology Program of China (No. NIMR-2013-9).
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