International Journal of Systematic and Evolutionary Microbiology (2014), 64, 968–972
DOI 10.1099/ijs.0.055004-0
Pontibacter yuliensis sp. nov., isolated from soil Hanjun Cao,4 Yao Nie,4 Xian-Chun Zeng, Linghua Xu, Zancan He, Xuesong Luo and Rina Wu Correspondence Xian-Chun Zeng [email protected]
State Key Laboratory of Biogeology and Environmental Geology & Department of Biological Science and Technology, School of Environmental Studies, China University of Geosciences (Wuhan), Wuhan 430074, PR China A Gram-staining-negative, rod-shaped and pink bacterium was isolated from the soil of a Populus euphratica forest in the Taklamakan desert, Xinjiang, China. It was designated strain H9XT. A 16S rRNA gene sequence homology search indicated that the isolate was most closely related to the family Cytophagaceae. The 16S rRNA gene of strain H9XT displayed 94.2–96.3 % sequence identities to those of type strains of other species of the genus Pontibacter. It only possessed menaquinone-7. The major cellular fatty acids of the novel isolate were iso-C15 : 0, C16 : 1v5c summed feature 3 (containing C16 : 1v6c and/or C16 : 1v7c) and summed feature 4 (comprising anteiso-C17 : 1 B and/or iso-C17 : 1 I). The major polar lipids were phosphatidylethanolamine, one unknown aminophospholipid, one unknown glycophospholipid and several unknown phospholipids. The DNA G+C content of this bacterium was 55.2 mol%. Based on the phenotypic and genotypic data presented, it can be concluded that this isolate represents a novel species of the genus Pontibacter, for which the name Pontibacter yuliensis sp. nov. is proposed. The type strain is H9XT (5CCTCC AB 2013047T5KCTC 32396T).
The genus Pontibacter, first described by Nedashkovskaya et al. (2005), is a member of the family Cytophagaceae. At the time of writing, 10 species with validly published names have been described, including Pontibacter actiniarum (Nedashkovskaya et al., 2005), Pontibacter roseus (Suresh et al., 2006; Wang et al., 2010), Pontibacter akesuensis (Zhou et al., 2007), Pontibacter korlensis (Zhang et al., 2008), Pontibacter xinjiangensis (Wang et al., 2010), Pontibacter niistensis (Dastager et al., 2010), Pontibacter populi (Xu et al., 2012), Pontibacter lucknowensis (Dwivedi et al., 2013), Pontibacter saemangeumensis (Kang et al., 2013) and Pontibacter ramchanderi (Singh et al., 2013). The genome of Pontibacter sp. BAB1700, of which the 16S rRNA gene showed 100 % sequence identity with P. lucknowensis DM9T, has recently been sequenced. This strain is a halotolerant bacterium and possesses gene clusters that are involved in vitamin biosynthesis or responsible for resistance to various metals and antibiotics (Joshi et al., 2012). This indicates that the bacteria belonging to this genus have evolved to adapt to some unusual or extreme environments.
Abbreviations: APL, aminophospholipid; GPL, glycophospholipid; PE, phosphatidylethanolamine. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain H9XT is KF146891. Two supplementary figures are available in the online Supplementary Material. 4These authors contributed equally to this article.
968
The Taklamakan desert is the world’s second largest shifting sand desert, where the environmental conditions are extremely rigorous. It was found that the Populus euphratica forest growing in the desert area has drastically degenerated in recent years. Our research has focused on exploration of the correlation between the functional microbial community changes and the forest degeneration in the desert. In order to isolate cultivable micro-organisms from the Populus euphratica forest area, we collected soil samples from the woods. The samples were suspended in 0.85 % (w/v) NaCl solution. After removal of insoluble sands and large particles, supernatant containing bacteria was serially diluted and plated onto on a modified marine broth agar [containing 15 g marine broth 2216 (Difco) and 15 g agar l21; pH 7.2]. Based on the colony morphology and Gram staining of the cells, approximately 40 different bacteria were isolated from 1 mg of soil. The genomic DNAs of these bacteria were isolated using MiniBEST Bacterial Genomic DNA Extraction kit ver.2.0 (TaKaRa). Fragments of the 16S rRNA genes were amplified by PCR using the primers 27F and 1492R as described previously (Lane, 1991). PCR products were purified and sequenced by GenScript. We thus obtained nearly full-length 16S rRNA gene sequences of all isolated bacteria. A search for nucleotide sequence homology and calculation of identities for 16S rRNA genes were achieved using NCBI BLAST and the EzTaxon-e server (http:// eztaxon-e.ezbiocloud.net/; Kim et al., 2012). The results indicated that we had obtained a novel bacterial strain that 055004 G 2014 IUMS Printed in Great Britain
Pontibacter yuliensis sp. nov.
was designated H9XT. The nucleotide sequence of H9XT displays 94.2–96.3 % identities with those of the species of the genus Pontibacter. Phylogenetic trees were reconstructed from the alignment of the 16S rRNA gene sequences of H9XT and those of closely related species of the genus Pontibacter using the neighbour-joining and maximum-likelihood methods (Tamura et al., 2011). The topologies of the phylogenetic trees were evaluated using the bootstrap resampling method with 1000 replicates. We found that the 16S rRNA gene of strain H9XT falls into a distinct evolutionary branch in the phylogenetic trees (Fig. 1 and Fig. S1 available in the online Supplementary Material). This indicates that the novel bacterium is likely to represent a novel species belonging to the genus Pontibacter. Strain H9XT formed circular, pink and smooth colonies on 0.36MB agar plate after incubation at 30 uC for four days. Morphology of the bacterial cells was observed under light and phase-contrast microscopy (BX51; Olympus). Gliding motility tests were performed by preparing a light suspension of the bacterial cells in water and then placing a drop on quarter-strength marine 2216 medium solidified with 1 % agarose. After 16 h of incubation at 20 uC, the inoculated area was covered with a glass coverslip and examined by oil immersion
100 93
0.02
phase-contrast microscopy (Bowman, 2000). Cells of strain H9XT were Gram-staining-negative, rod-shaped and motile by gliding. Bathochromatic shift assay was employed to determine whether the flexirubin-type pigments were present in the cells or not. A 20 % (w/v) KOH solution was used for the test (Fautz & Reichenbach, 1980). We found that flexirubin-type pigments were not detectable in the cells. NaCl tolerance was determined by growing the bacteria in 0.16TSB medium (Difco) containing different concentrations of NaCl (0–10 %, w/v), respectively. The bacterial growth was also examined at different temperatures (4, 10, 20, 28, 30, 35, 37, 42 uC) and at different pH values (pH 5.0– 11.0, 1.0 pH increments), respectively. Oxidase activity was evaluated via the oxidation of 1 % p-aminodimethylaniline oxalate. Catalase activity was determined by measurements of bubble production after the addition of 3 % (v/v) hydrogen peroxide solution. The bacterium was also tested for hydrolysis of starch (1 %, w/v), cellulose (0.1 %, w/v), chitin from crab shells (1 %, w/v), casein (1 %, w/v) and tyrosine (0.5 %, w/v) as described by Smibert &Krieg (1994). Other enzyme activities and biochemical features were determined using API kits (API 20NE, API 20E, API
Pontibacter ramchanderi LP43T (JQ806111) Pontibacter lucknowensis DM9T (JN561788) ‘Pontibacter salisaro’ HMC5104 (FJ903180) Pontibacter populi HLY7-15T (HQ223078) ‘Pontibacter rhizosphera’ IMTB-1969 (HQ534295) Pontibacter niistensis NII-0905T (FJ897494)
Pontibacter korlensis X14-1T (DQ888330) Pontibacter roseus SRC-1T (AM049256) Pontibacter actiniarum KMM 6156T (AY989908) Pontibacter akesuensis AKS 1T (DQ672723) 100
Pontibacter saemangeumensis GCM0142T (JN607163) 91
Pontibacter xinjiangensis 311-10T (FJ004994) Pontibacter yuliensis H9XT (KF146891) Adhaeribacter aquaticus MBRG1.5T (AJ626894)
72
Hymenobacter roseosalivarius AA-718T (Y18833) Fontibacter flavus CC-GZM-130T (FN421478)
Fig. 1. Neighbour-joining phylogenetic tree based on nucleotide sequence alignment of 16S rRNA genes of the isolate Pontibacter yuliensis sp. nov. H9XT and other closely related strains. Bootstrap values (expressed as percentages of 1000 replicates) that are .70 % are shown at branch points. Bar, 0.02 substitutions per nucleotide position. http://ijs.sgmjournals.org
969
H. Cao and others
50CH and API ZYM) according to the manufacturer’s instruction (bioMe´rieux). This bacterium showed many phenotypic features consistent with those of the species of
the genus Pontibacter. For example, it was positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase,
Table 1. Differential phenotypic characteristics of strain H9XT and related type strains of species of the genus Pontibacter Strains: 1, H9XT; 2, P. korlensis CCTCC AB 206081T; 3, P. actiniarum KMM 6156T; 4, P. xinjiangensis CCTCC AB 207200T; 5, P. saemangeumensis GCM0142T. All data were obtained from this study, except for those for strains 2–5 indicated with asterisks which were taken from Zhang et al. (2008), Nedashkovskaya et al. (2005), Wang et al. (2010) and Kang et al. (2013), respectively. All strains are positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase and negative for indole and H2S production, glucose fermentation, Voges–Proskauer test, arginine dihydrolase, urease, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase and a-fucosidase. +, Positive; 2, negative; W, weakly positive; ND, not determined. Characteristic Temperature range (uC) pH Salinity (NaCl concentration, %) Motility Oxidase Nitrate reduction Hydrolysis of: Starch Casein Gelatin Aesculin ONPG Assimilation (20NE/32GN) Citrate D-Glucose D-Mannose N-Acetylglucosamine Maltose Gluconate Capric acid L-Rhamnose Inositol Sucrose Suberic acid Malonate L-Alanine 5-Ketogluconate Glycogen Melibiose L-Histidine 2-Ketogluconate 3-Hydroxybutyric acid API ZYM (enzymes) Lipase (C14) Cystine arylamidase Trypsin a-Chymotrypsin a-Galactosidase b-Galactosidase b-Glucuronidase a-Glucosidase b-Glucosidase N-Acetyl-b-glucosaminidase a-Mannosidase
970
1
2
3
4
5*
4–37 6.0–10.0 0–7.5 +
6–43*
2
7–45* 5.5–11.0* 0–8* + 2 2
4–37* 6.0–10.0* 0–5* 2 2 +
5–30 6.0–10.0 0–2 2 + 2
+ + 2 +
+ 2 + +
+ + + +
W
W
2 2 + 2 2
+ + 2 + +
2 2 2 2 2 2
+ + + +
W
2
2 2 2 + 2 2 2 2 2
W
2 2
+ + + + + + +
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
2 2 2 2 2 2 2 2 2 2 2
+ + 2 2 + + + + + + 2
2 + + 2 2 2 2 2 2 + 2
2 + + + + + 2 2 2 + 2
W
W
W
W
+ + + W
ND
0–10* + 2 2
W
2 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
2 2 + 2 + + 2 + + + +
International Journal of Systematic and Evolutionary Microbiology 64
Pontibacter yuliensis sp. nov.
naphthol-AS-BI-phosphohydrolase and negative for indole and H2S production, glucose fermentation, Voges– Proskauer test, arginine dihydrolase, urease, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase and a-fucosidase. Physiological features, which distinguish the novel isolate from closely related species, are shown in Table 1. This strongly indicates that this isolate is a representative of a novel species of the genus Pontibacter. Respiratory quinones were extracted and detected by HPLC as described previously (Komagata & Suzuki, 1987). Menaquinone-7 (MK-7) was the only menaquinone in the cells. The presence of this chemotaxonomic marker is consistent with the characteristics of the species of the genus Pontibacter. DNA G+C content of strain H9XT was determined using HPLC (UltiMate 3000; Dionex) (Mesbah et al., 1989). The DNA G+C content was 55.2 mol%. Polar lipids were isolated using the standard TLC technique (Minnikin et al., 1984). Phosphatidylethanolamine (PE) was found to be the major polar lipid in the cells. One aminophospholipid (APL) and one glycophospholipid (GPL) were also readily detected. In addition, we also found several unidentified phospholipids (Fig. S2). Comparing the available data, we found that the polar lipids of different species in this genus are highly divergent, and all these species possess PE as one of the major polar lipids. For analysis of fatty acid methyl esters (FAMEs), the novel isolate and closely related type strains of species of the genus Pontibacter were cultured on modified marine broth agar for appropriate time. FAMEs were then prepared and analysed using the Sherlock Microbial Identification System (MIDI). We showed that its fatty acids, which contain iso-C15 : 0 (19.0 %), C16 : 1v5c (11.1 %), summed feature 3 (containing C16 : 1v6c and/or C16 : 1v7c) (20.1 %) and summed feature 4 (comprising anteiso-C17 : 1 B and/or iso-C17 : 1 I) (20.5 %) as the major constituents, are similar to those of the species of the genus Pontibacter. Comparisons of fatty acid compositions of H9XT with those of the closely related species are shown in Table 2. Based on the phenotypic and genotypic data presented here, it is concluded that strain H9XT represents a novel species of the genus Pontibacter, for which the name Pontibacter yuliensis sp. nov. is proposed. Description of Pontibacter yuliensis sp. nov. Pontibacter yuliensis (yu.li.en9sis. N.L. masc. adj. yuliensis pertaining to Yuli, a county of Xinjiang from where the type strain was isolated). Cells are Gram-staining-negative, aerobic, rod-shaped (0.4–0.661.2–2.0 mm) and motile by gliding. Colonies on 0.36MB are circular, convex, pink and smooth. Growth occurs at 4–37 uC (optimum 30 uC) and pH 6.0– 10.0. NaCl concentrations for growth are at 0–7.5 % (w/v). The bacterium is oxidase positive and catalase positive. It can hydrolyse casein, aesculin, starch and ONPG, but not tyrosine, chitin, gelatin or cellulose. Nitrate reduction, H2S http://ijs.sgmjournals.org
Table 2. Fatty acid profiles (%) of strain H9XT and related type strains of species of the genus Pontibacter Strains: 1, H9XT; 2, P. korlensis CCTCC AB 206081T; 3, P. actiniarum KMM 6156T; 4, P. xinjiangensis CCTCC AB 207200T; 5, P. saemangeumensis GCM0142T. All data were collected during this study except those for strain 5 which were taken from Kang et al. (2013). TR, Trace (,1 %). Fatty acid iso-C13 : 0 3-OH Summed feature iso-C15 : 0 anteiso-C15 : 0 C15 : 1v6c iso-C16 : 1 H iso-C16 : 0 Summed feature C16 : 1v5c C16 : 0 iso-C15 : 0 3-OH iso-C15 : 1 F Summed feature Summed feature iso-C17 : 0 anteiso-C17 : 0 C17 : 0 C17 : 1v6c Summed feature Summed feature C18 : 0 iso-C17 : 0 3-OH iso-C18 : 1 H iso-C19 : 1 I C20 : 0
1
2
1.1 19.0
TR
3 TR
1
14.8
TR
TR
TR
TR
3
20.1 11.1 5.3 1.8
7.5 6.5 2.9 3.8
10.2 3.1 1.8 3.5
23.0 10.4 2.1 2.7
9.3 1.7 1.0
TR
5
1.3
1.5 35.1
TR
2.3 1.6
4
22.3 2.5 2.9 3.5 1.1 6.2 4.1 TR
1.2
TR
9 4
TR
20.5 TR
31.5 5.6
25.8 3.7
1.5 26.4 1.4
36.8 1.5
TR TR
5 8
7.1 3.2
3.5 1.4
1.4 1.3
TR
6.4
1.0 10.5
1.2 8.6
2.1 1.3 1.9 1.8 7.6
4.3 TR TR TR
3.9
TR
3.1 1.1
Summed feature 1: iso-C15 : 1 H/C13 : 0 3-OH; summed feature 3: C16 : 1v7c/C16 : 1v6c; summed feature 4: iso-C17 : 1 I/anteiso-C17 : 1 B; summed feature 5: C18 : 2v6,9c/anteiso-C18 : 0; summed feature 8: C18 : 1v7c/C18 : 1v6c; Summed feature 9: iso-C17 : 1v9c/10-methyl C16 : 0.
production, citrate utilization, indole production, glucose acidification, arginine dihydrolase and urease are negative. It assimilates suberic acid and L-Histidine, but not Dglucose, L-arabinose, D-mannose, D-mannitol, N-acetylglucosamine, maltose, gluconate, adipate, malate, trisodium citrate, phenylacetic acid, L-rhamnose, D-ribose, inositol, sucrose, malonate, lactate, L-alanine, 5-ketogluconate, glycogen, melibiose, 2-ketogluconate and 3-hydroxybutyric acid. The type strain is positive for the activities of alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase and negative for the activities of lipase (C14), cystine arylamidase, trypsin, achymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase. The major fatty 971
H. Cao and others
acids are iso-C15 : 0, C16 : 1v5c, summed feature 3 (containing C16 : 1v6c and/or C16 : 1v7c) and summed feature 4 (comprising anteiso-C17 : 1B and/or iso-C17 : 1I). MK-7 is the predominant menaquinone. The major polar lipids are PE, APL, GPL and several unknown phospholipids.
Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid Techniques
in Bacterial Systematics, pp. 115–176. Edited by E. Stackebrandt & M. Goodfellow. Chichester: Wiley. Mesbah, M., Premachandran, U. & Whitman, W. (1989). Precise
measurement of the G+C content of desoxyribonucleic acids by high performance liquid chromatography. Int Syst Bacteriol 39, 159–167.
The type strain, H9XT (5CCTCC AB 2013047T5KCTC 32396T), was isolated from the soil of Populus euphratica forest in Yuli County. The DNA G+C content is 55.2 mol%.
Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated
Acknowledgements
Nedashkovskaya, O. I., Kim, S. B., Suzuki, M., Shevchenko, L. S., Lee, M. S., Lee, K. H., Park, M. S., Frolova, G. M., Oh, H. W. & other authors (2005). Pontibacter actiniarum gen. nov., sp. nov., a
This work was supported by the National Natural Science Foundation of China (grant no. 41272257 and no. 41072181) and the Research Fund for the Faculty of State Key Laboratory of Biogeology and Environmental Geology (BGEG) (grant no. GBL11208) granted to Prof. Xian-Chun Zeng.
procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241.
novel member of the phylum ‘Bacteroidetes’, and proposal of Reichenbachiella gen. nov. as a replacement for the illegitimate prokaryotic generic name Reichenbachia Nedashkovskaya et al. 2003. Int J Syst Evol Microbiol 55, 2583–2588. Singh, A. K., Garg, N., Sangwan, N., Negi, V., Kumar, R., Vikram, S. & Lal, R. (2013). Pontibacter ramchanderi sp. nov., isolated from
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gen. nov., sp. nov., isolated from muddy water, belonging to the family ‘‘Flexibacteraceae’’. Int J Syst Evol Microbiol 56, 1703–1707. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using
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International Journal of Systematic and Evolutionary Microbiology 64
DOI 10.1099/ijs.0.055004-0
Pontibacter yuliensis sp. nov., isolated from soil Hanjun Cao,4 Yao Nie,4 Xian-Chun Zeng, Linghua Xu, Zancan He, Xuesong Luo and Rina Wu Correspondence Xian-Chun Zeng [email protected]
State Key Laboratory of Biogeology and Environmental Geology & Department of Biological Science and Technology, School of Environmental Studies, China University of Geosciences (Wuhan), Wuhan 430074, PR China A Gram-staining-negative, rod-shaped and pink bacterium was isolated from the soil of a Populus euphratica forest in the Taklamakan desert, Xinjiang, China. It was designated strain H9XT. A 16S rRNA gene sequence homology search indicated that the isolate was most closely related to the family Cytophagaceae. The 16S rRNA gene of strain H9XT displayed 94.2–96.3 % sequence identities to those of type strains of other species of the genus Pontibacter. It only possessed menaquinone-7. The major cellular fatty acids of the novel isolate were iso-C15 : 0, C16 : 1v5c summed feature 3 (containing C16 : 1v6c and/or C16 : 1v7c) and summed feature 4 (comprising anteiso-C17 : 1 B and/or iso-C17 : 1 I). The major polar lipids were phosphatidylethanolamine, one unknown aminophospholipid, one unknown glycophospholipid and several unknown phospholipids. The DNA G+C content of this bacterium was 55.2 mol%. Based on the phenotypic and genotypic data presented, it can be concluded that this isolate represents a novel species of the genus Pontibacter, for which the name Pontibacter yuliensis sp. nov. is proposed. The type strain is H9XT (5CCTCC AB 2013047T5KCTC 32396T).
The genus Pontibacter, first described by Nedashkovskaya et al. (2005), is a member of the family Cytophagaceae. At the time of writing, 10 species with validly published names have been described, including Pontibacter actiniarum (Nedashkovskaya et al., 2005), Pontibacter roseus (Suresh et al., 2006; Wang et al., 2010), Pontibacter akesuensis (Zhou et al., 2007), Pontibacter korlensis (Zhang et al., 2008), Pontibacter xinjiangensis (Wang et al., 2010), Pontibacter niistensis (Dastager et al., 2010), Pontibacter populi (Xu et al., 2012), Pontibacter lucknowensis (Dwivedi et al., 2013), Pontibacter saemangeumensis (Kang et al., 2013) and Pontibacter ramchanderi (Singh et al., 2013). The genome of Pontibacter sp. BAB1700, of which the 16S rRNA gene showed 100 % sequence identity with P. lucknowensis DM9T, has recently been sequenced. This strain is a halotolerant bacterium and possesses gene clusters that are involved in vitamin biosynthesis or responsible for resistance to various metals and antibiotics (Joshi et al., 2012). This indicates that the bacteria belonging to this genus have evolved to adapt to some unusual or extreme environments.
Abbreviations: APL, aminophospholipid; GPL, glycophospholipid; PE, phosphatidylethanolamine. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain H9XT is KF146891. Two supplementary figures are available in the online Supplementary Material. 4These authors contributed equally to this article.
968
The Taklamakan desert is the world’s second largest shifting sand desert, where the environmental conditions are extremely rigorous. It was found that the Populus euphratica forest growing in the desert area has drastically degenerated in recent years. Our research has focused on exploration of the correlation between the functional microbial community changes and the forest degeneration in the desert. In order to isolate cultivable micro-organisms from the Populus euphratica forest area, we collected soil samples from the woods. The samples were suspended in 0.85 % (w/v) NaCl solution. After removal of insoluble sands and large particles, supernatant containing bacteria was serially diluted and plated onto on a modified marine broth agar [containing 15 g marine broth 2216 (Difco) and 15 g agar l21; pH 7.2]. Based on the colony morphology and Gram staining of the cells, approximately 40 different bacteria were isolated from 1 mg of soil. The genomic DNAs of these bacteria were isolated using MiniBEST Bacterial Genomic DNA Extraction kit ver.2.0 (TaKaRa). Fragments of the 16S rRNA genes were amplified by PCR using the primers 27F and 1492R as described previously (Lane, 1991). PCR products were purified and sequenced by GenScript. We thus obtained nearly full-length 16S rRNA gene sequences of all isolated bacteria. A search for nucleotide sequence homology and calculation of identities for 16S rRNA genes were achieved using NCBI BLAST and the EzTaxon-e server (http:// eztaxon-e.ezbiocloud.net/; Kim et al., 2012). The results indicated that we had obtained a novel bacterial strain that 055004 G 2014 IUMS Printed in Great Britain
Pontibacter yuliensis sp. nov.
was designated H9XT. The nucleotide sequence of H9XT displays 94.2–96.3 % identities with those of the species of the genus Pontibacter. Phylogenetic trees were reconstructed from the alignment of the 16S rRNA gene sequences of H9XT and those of closely related species of the genus Pontibacter using the neighbour-joining and maximum-likelihood methods (Tamura et al., 2011). The topologies of the phylogenetic trees were evaluated using the bootstrap resampling method with 1000 replicates. We found that the 16S rRNA gene of strain H9XT falls into a distinct evolutionary branch in the phylogenetic trees (Fig. 1 and Fig. S1 available in the online Supplementary Material). This indicates that the novel bacterium is likely to represent a novel species belonging to the genus Pontibacter. Strain H9XT formed circular, pink and smooth colonies on 0.36MB agar plate after incubation at 30 uC for four days. Morphology of the bacterial cells was observed under light and phase-contrast microscopy (BX51; Olympus). Gliding motility tests were performed by preparing a light suspension of the bacterial cells in water and then placing a drop on quarter-strength marine 2216 medium solidified with 1 % agarose. After 16 h of incubation at 20 uC, the inoculated area was covered with a glass coverslip and examined by oil immersion
100 93
0.02
phase-contrast microscopy (Bowman, 2000). Cells of strain H9XT were Gram-staining-negative, rod-shaped and motile by gliding. Bathochromatic shift assay was employed to determine whether the flexirubin-type pigments were present in the cells or not. A 20 % (w/v) KOH solution was used for the test (Fautz & Reichenbach, 1980). We found that flexirubin-type pigments were not detectable in the cells. NaCl tolerance was determined by growing the bacteria in 0.16TSB medium (Difco) containing different concentrations of NaCl (0–10 %, w/v), respectively. The bacterial growth was also examined at different temperatures (4, 10, 20, 28, 30, 35, 37, 42 uC) and at different pH values (pH 5.0– 11.0, 1.0 pH increments), respectively. Oxidase activity was evaluated via the oxidation of 1 % p-aminodimethylaniline oxalate. Catalase activity was determined by measurements of bubble production after the addition of 3 % (v/v) hydrogen peroxide solution. The bacterium was also tested for hydrolysis of starch (1 %, w/v), cellulose (0.1 %, w/v), chitin from crab shells (1 %, w/v), casein (1 %, w/v) and tyrosine (0.5 %, w/v) as described by Smibert &Krieg (1994). Other enzyme activities and biochemical features were determined using API kits (API 20NE, API 20E, API
Pontibacter ramchanderi LP43T (JQ806111) Pontibacter lucknowensis DM9T (JN561788) ‘Pontibacter salisaro’ HMC5104 (FJ903180) Pontibacter populi HLY7-15T (HQ223078) ‘Pontibacter rhizosphera’ IMTB-1969 (HQ534295) Pontibacter niistensis NII-0905T (FJ897494)
Pontibacter korlensis X14-1T (DQ888330) Pontibacter roseus SRC-1T (AM049256) Pontibacter actiniarum KMM 6156T (AY989908) Pontibacter akesuensis AKS 1T (DQ672723) 100
Pontibacter saemangeumensis GCM0142T (JN607163) 91
Pontibacter xinjiangensis 311-10T (FJ004994) Pontibacter yuliensis H9XT (KF146891) Adhaeribacter aquaticus MBRG1.5T (AJ626894)
72
Hymenobacter roseosalivarius AA-718T (Y18833) Fontibacter flavus CC-GZM-130T (FN421478)
Fig. 1. Neighbour-joining phylogenetic tree based on nucleotide sequence alignment of 16S rRNA genes of the isolate Pontibacter yuliensis sp. nov. H9XT and other closely related strains. Bootstrap values (expressed as percentages of 1000 replicates) that are .70 % are shown at branch points. Bar, 0.02 substitutions per nucleotide position. http://ijs.sgmjournals.org
969
H. Cao and others
50CH and API ZYM) according to the manufacturer’s instruction (bioMe´rieux). This bacterium showed many phenotypic features consistent with those of the species of
the genus Pontibacter. For example, it was positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase,
Table 1. Differential phenotypic characteristics of strain H9XT and related type strains of species of the genus Pontibacter Strains: 1, H9XT; 2, P. korlensis CCTCC AB 206081T; 3, P. actiniarum KMM 6156T; 4, P. xinjiangensis CCTCC AB 207200T; 5, P. saemangeumensis GCM0142T. All data were obtained from this study, except for those for strains 2–5 indicated with asterisks which were taken from Zhang et al. (2008), Nedashkovskaya et al. (2005), Wang et al. (2010) and Kang et al. (2013), respectively. All strains are positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase and negative for indole and H2S production, glucose fermentation, Voges–Proskauer test, arginine dihydrolase, urease, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase and a-fucosidase. +, Positive; 2, negative; W, weakly positive; ND, not determined. Characteristic Temperature range (uC) pH Salinity (NaCl concentration, %) Motility Oxidase Nitrate reduction Hydrolysis of: Starch Casein Gelatin Aesculin ONPG Assimilation (20NE/32GN) Citrate D-Glucose D-Mannose N-Acetylglucosamine Maltose Gluconate Capric acid L-Rhamnose Inositol Sucrose Suberic acid Malonate L-Alanine 5-Ketogluconate Glycogen Melibiose L-Histidine 2-Ketogluconate 3-Hydroxybutyric acid API ZYM (enzymes) Lipase (C14) Cystine arylamidase Trypsin a-Chymotrypsin a-Galactosidase b-Galactosidase b-Glucuronidase a-Glucosidase b-Glucosidase N-Acetyl-b-glucosaminidase a-Mannosidase
970
1
2
3
4
5*
4–37 6.0–10.0 0–7.5 +
6–43*
2
7–45* 5.5–11.0* 0–8* + 2 2
4–37* 6.0–10.0* 0–5* 2 2 +
5–30 6.0–10.0 0–2 2 + 2
+ + 2 +
+ 2 + +
+ + + +
W
W
2 2 + 2 2
+ + 2 + +
2 2 2 2 2 2
+ + + +
W
2
2 2 2 + 2 2 2 2 2
W
2 2
+ + + + + + +
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
2 2 2 2 2 2 2 2 2 2 2
+ + 2 2 + + + + + + 2
2 + + 2 2 2 2 2 2 + 2
2 + + + + + 2 2 2 + 2
W
W
W
W
+ + + W
ND
0–10* + 2 2
W
2 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
2 2 + 2 + + 2 + + + +
International Journal of Systematic and Evolutionary Microbiology 64
Pontibacter yuliensis sp. nov.
naphthol-AS-BI-phosphohydrolase and negative for indole and H2S production, glucose fermentation, Voges– Proskauer test, arginine dihydrolase, urease, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase and a-fucosidase. Physiological features, which distinguish the novel isolate from closely related species, are shown in Table 1. This strongly indicates that this isolate is a representative of a novel species of the genus Pontibacter. Respiratory quinones were extracted and detected by HPLC as described previously (Komagata & Suzuki, 1987). Menaquinone-7 (MK-7) was the only menaquinone in the cells. The presence of this chemotaxonomic marker is consistent with the characteristics of the species of the genus Pontibacter. DNA G+C content of strain H9XT was determined using HPLC (UltiMate 3000; Dionex) (Mesbah et al., 1989). The DNA G+C content was 55.2 mol%. Polar lipids were isolated using the standard TLC technique (Minnikin et al., 1984). Phosphatidylethanolamine (PE) was found to be the major polar lipid in the cells. One aminophospholipid (APL) and one glycophospholipid (GPL) were also readily detected. In addition, we also found several unidentified phospholipids (Fig. S2). Comparing the available data, we found that the polar lipids of different species in this genus are highly divergent, and all these species possess PE as one of the major polar lipids. For analysis of fatty acid methyl esters (FAMEs), the novel isolate and closely related type strains of species of the genus Pontibacter were cultured on modified marine broth agar for appropriate time. FAMEs were then prepared and analysed using the Sherlock Microbial Identification System (MIDI). We showed that its fatty acids, which contain iso-C15 : 0 (19.0 %), C16 : 1v5c (11.1 %), summed feature 3 (containing C16 : 1v6c and/or C16 : 1v7c) (20.1 %) and summed feature 4 (comprising anteiso-C17 : 1 B and/or iso-C17 : 1 I) (20.5 %) as the major constituents, are similar to those of the species of the genus Pontibacter. Comparisons of fatty acid compositions of H9XT with those of the closely related species are shown in Table 2. Based on the phenotypic and genotypic data presented here, it is concluded that strain H9XT represents a novel species of the genus Pontibacter, for which the name Pontibacter yuliensis sp. nov. is proposed. Description of Pontibacter yuliensis sp. nov. Pontibacter yuliensis (yu.li.en9sis. N.L. masc. adj. yuliensis pertaining to Yuli, a county of Xinjiang from where the type strain was isolated). Cells are Gram-staining-negative, aerobic, rod-shaped (0.4–0.661.2–2.0 mm) and motile by gliding. Colonies on 0.36MB are circular, convex, pink and smooth. Growth occurs at 4–37 uC (optimum 30 uC) and pH 6.0– 10.0. NaCl concentrations for growth are at 0–7.5 % (w/v). The bacterium is oxidase positive and catalase positive. It can hydrolyse casein, aesculin, starch and ONPG, but not tyrosine, chitin, gelatin or cellulose. Nitrate reduction, H2S http://ijs.sgmjournals.org
Table 2. Fatty acid profiles (%) of strain H9XT and related type strains of species of the genus Pontibacter Strains: 1, H9XT; 2, P. korlensis CCTCC AB 206081T; 3, P. actiniarum KMM 6156T; 4, P. xinjiangensis CCTCC AB 207200T; 5, P. saemangeumensis GCM0142T. All data were collected during this study except those for strain 5 which were taken from Kang et al. (2013). TR, Trace (,1 %). Fatty acid iso-C13 : 0 3-OH Summed feature iso-C15 : 0 anteiso-C15 : 0 C15 : 1v6c iso-C16 : 1 H iso-C16 : 0 Summed feature C16 : 1v5c C16 : 0 iso-C15 : 0 3-OH iso-C15 : 1 F Summed feature Summed feature iso-C17 : 0 anteiso-C17 : 0 C17 : 0 C17 : 1v6c Summed feature Summed feature C18 : 0 iso-C17 : 0 3-OH iso-C18 : 1 H iso-C19 : 1 I C20 : 0
1
2
1.1 19.0
TR
3 TR
1
14.8
TR
TR
TR
TR
3
20.1 11.1 5.3 1.8
7.5 6.5 2.9 3.8
10.2 3.1 1.8 3.5
23.0 10.4 2.1 2.7
9.3 1.7 1.0
TR
5
1.3
1.5 35.1
TR
2.3 1.6
4
22.3 2.5 2.9 3.5 1.1 6.2 4.1 TR
1.2
TR
9 4
TR
20.5 TR
31.5 5.6
25.8 3.7
1.5 26.4 1.4
36.8 1.5
TR TR
5 8
7.1 3.2
3.5 1.4
1.4 1.3
TR
6.4
1.0 10.5
1.2 8.6
2.1 1.3 1.9 1.8 7.6
4.3 TR TR TR
3.9
TR
3.1 1.1
Summed feature 1: iso-C15 : 1 H/C13 : 0 3-OH; summed feature 3: C16 : 1v7c/C16 : 1v6c; summed feature 4: iso-C17 : 1 I/anteiso-C17 : 1 B; summed feature 5: C18 : 2v6,9c/anteiso-C18 : 0; summed feature 8: C18 : 1v7c/C18 : 1v6c; Summed feature 9: iso-C17 : 1v9c/10-methyl C16 : 0.
production, citrate utilization, indole production, glucose acidification, arginine dihydrolase and urease are negative. It assimilates suberic acid and L-Histidine, but not Dglucose, L-arabinose, D-mannose, D-mannitol, N-acetylglucosamine, maltose, gluconate, adipate, malate, trisodium citrate, phenylacetic acid, L-rhamnose, D-ribose, inositol, sucrose, malonate, lactate, L-alanine, 5-ketogluconate, glycogen, melibiose, 2-ketogluconate and 3-hydroxybutyric acid. The type strain is positive for the activities of alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase and negative for the activities of lipase (C14), cystine arylamidase, trypsin, achymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase. The major fatty 971
H. Cao and others
acids are iso-C15 : 0, C16 : 1v5c, summed feature 3 (containing C16 : 1v6c and/or C16 : 1v7c) and summed feature 4 (comprising anteiso-C17 : 1B and/or iso-C17 : 1I). MK-7 is the predominant menaquinone. The major polar lipids are PE, APL, GPL and several unknown phospholipids.
Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid Techniques
in Bacterial Systematics, pp. 115–176. Edited by E. Stackebrandt & M. Goodfellow. Chichester: Wiley. Mesbah, M., Premachandran, U. & Whitman, W. (1989). Precise
measurement of the G+C content of desoxyribonucleic acids by high performance liquid chromatography. Int Syst Bacteriol 39, 159–167.
The type strain, H9XT (5CCTCC AB 2013047T5KCTC 32396T), was isolated from the soil of Populus euphratica forest in Yuli County. The DNA G+C content is 55.2 mol%.
Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated
Acknowledgements
Nedashkovskaya, O. I., Kim, S. B., Suzuki, M., Shevchenko, L. S., Lee, M. S., Lee, K. H., Park, M. S., Frolova, G. M., Oh, H. W. & other authors (2005). Pontibacter actiniarum gen. nov., sp. nov., a
This work was supported by the National Natural Science Foundation of China (grant no. 41272257 and no. 41072181) and the Research Fund for the Faculty of State Key Laboratory of Biogeology and Environmental Geology (BGEG) (grant no. GBL11208) granted to Prof. Xian-Chun Zeng.
procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241.
novel member of the phylum ‘Bacteroidetes’, and proposal of Reichenbachiella gen. nov. as a replacement for the illegitimate prokaryotic generic name Reichenbachia Nedashkovskaya et al. 2003. Int J Syst Evol Microbiol 55, 2583–2588. Singh, A. K., Garg, N., Sangwan, N., Negi, V., Kumar, R., Vikram, S. & Lal, R. (2013). Pontibacter ramchanderi sp. nov., isolated from
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