International Journal of Systematic and Evolutionary Microbiology (2015), 65, 214–219
DOI 10.1099/ijs.0.060996-0
Crenobacter luteus gen. nov., sp. nov., isolated from a hot spring Lei Dong,13 Hong Ming,1,23 En-Min Zhou,1 Yi-Rui Yin,1 Lan Liu,1 Hui-Geng Feng,2 Wen-Dong Xian,1 Guo-Xing Nie3 and Wen-Jun Li1,4 Correspondence Wen-Jun Li [email protected] or [email protected]
1
Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education, Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, PR China
2
Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang 453003, PR China
3
College of Fisheries, Henan Normal University, Xinxiang 453007, PR China
4
State Key Laboratory of Biocontrol, Key Laboratory of Biodiversity Dynamics and Conservation of Guangdong Higher Education Institutes, College of Ecology and Evolution, Sun Yat-Sen University, Guangzhou 510275, PR China
A slightly thermophilic, Gram-staining-negative and strictly aerobic bacteria, designated strain YIM 78141T, was isolated from a sediment sample collected at Hehua hot spring, Tengchong, Yunnan province, south-west China. Cells of the strain were short-rod-shaped and colonies were yellowish and circular. The strain grew at pH 6.0–10.0 (optimum, pH 8.0–9.0) and 10–55 6C (optimum, 40–50 6C). Phylogenetic analyses based on 16S rRNA gene sequence comparison demonstrated that strain YIM 78141T belongs to the family Neisseriaceae, and strain YIM 78141T also showed low levels of 16S rRNA gene sequence similarity (below 93.4 %) with all other genera in this family. The only quinone was ubiquinone 8 and the genomic DNA G+C content was 67.3 mol%. Major fatty acids (.5 %) were C12 : 0, C16 : 0, C18 : 1v7c and summed feature 3. The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, phospholipids of unknown structure containing aminoglycophospholipid and three unidentified polar lipids. On the basis of the morphological, physiological and biochemical characteristics as well as genotypic data, this strain should be classified as a representative of a novel genus and species of the family Neisseriaceae, for which the name Crenobacter luteus gen. nov., sp. nov. is proposed. The type strain is YIM 78141T (5BCRC 80650T5KCTC 32558T5 DSM 27258T).
The family Neisseriaceae belongs to the order Neisseriales and comprises 33 genera (http://www.bacterio.net/ -classifgenerafamilies.html#Neisseriaceae). In this family, the major distinguishing features among genera are biochemical characteristics, such as the production of arginine dihydrolase, glucose fermentation and G+C content of the genomic DNA, also supported by low 16S rRNA gene sequence similarities (Grimes et al., 1997; Yuen et al., 2001; Chern et al., 2004; Lau et al., 2006). Members of some of these genera, such as Laribacter and Microvirgula (Yuen et al., 2001; Patureau et al., 1998), do not have the ability to oxidize, ferment or assimilate sugars and have been referred to as asaccharolytic (Yuen et al., 2001). Members 3These authors contributed equally to this work. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain YIM 78141T is KF771276. Four supplementary figures are available with the online Supplementary Material.
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of these genera have been isolated from various environments, such as soil, water and clinical specimens (Patureau et al., 1998; Yuen et al., 2001; Vela et al., 2005; Lau et al., 2006). During an investigation of thermophilic microbial resources at Hehua hot spring in Tengchong, Yunnan province, southwest China, a slightly thermophilic strain of the family Neisseriaceae, designated YIM 78141T, was isolated from a hot spring sediment sample (24.9728u N 98.39706u E) by using a serial dilution technique. After 1 week of incubation on R2A agar medium (Becton Dickinson, BD) at 45 uC, the colony was picked and restreaked repeatedly onto R2A agar medium at 45 uC, until purity was confirmed. The purified strain was routinely cultivated and stored as aqueous glycerol suspensions (20 %, v/v) at 280 uC. Extraction of genomic DNA and PCR amplification of the 16S rRNA gene sequence were performed as described previously (Cui et al., 2001; Li et al., 2007). The resulting 060996 G 2015 IUMS Printed in Great Britain
Crenobacter luteus gen. nov., sp. nov.
16S rRNA gene sequence was compared with available 16S rRNA gene sequences of cultured species from GenBank via the BLAST program and the EzTaxon-e server (http:// eztaxon-e.ezbiocloud.net/; Kim et al., 2012). Phylogenetic analyses were performed using three tree-making algorithms: neighbour-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981) and maximumparsimony (Fitch, 1971), by using the software package MEGA version 5.0 (Tamura et al., 2011). Kimura’s twoparameter model was used to calculate evolutionary distance matrices of the neighbour-joining method and maximum-likelihood method (Kimura, 1980). The topology of the phylogenetic trees was evaluated by the bootstrap resampling method of Felsenstein (1985) with 1000 resamplings.
by using EzTaxon-e (Kim et al., 2012) indicated that strain YIM 78141T belongs to the family Neisseriaceae of the class Betaproteobacteria; sequence similarities among strain YIM 78141T and the type strains of all other species studied were less than 93.4 %. YIM 78141T was grouped into a larger, compact cluster (59 % bootstrap support) that was composed of the genera Gulbenkiania, Pseudogulbenkiania, Paludibacterium, Aquitalea, Chromobacterium, Aquaspirillum, Microvirgula and Laribacter (Fig. 1). Similar results were obtained using the maximum-parsimony and maximum-likelihood algorithms (Figs S1 and S2, available in the online Supplementary Material). The relatively high sequence divergence values (.6.6 %) showed that the novel isolate was distantly related to the described genera in the family Neisseriaceae.
An almost complete 16S rRNA gene sequence (1557 bp) of strain YIM 78141T was derived, and phylogenetic analysis
Biomass used for chemical studies was obtained from cultures grown on R2A agar medium at 45 uC for 3 days
100* Chromobacterium vaccinii MWU205T (JN120869) Chromobacterium piscinae CCM 3329T (AJ871127) 61
0.01
99
Chromobacterium violaceum ATCC 12472T (AE016825) 100* Chromobacterium pseudoviolaceum CCM 2076T (AJ871128)
93*
Chromobacterium subtsugae PRAA4-1T (AY344056) Chromobacterium aquaticum CC-SEYA-1T (EU109734)
95*
100*
Aquitalea magnusonii TRO-001DR8T (DQ018117)
54
100
Aquitalea denitrificans 5YN1-3T (EU594330) Gulbenkiania indica HT27T (DQ415656)
66* 53*
Chromobacterium haemolyticum MDA0585T (DQ785104)
100 Gulbenkiania mobilis E4FC31T (AM295491) Paludibacterium yongneupense 5YN8-15T (AM396358) Pseudogulbenkiania subflava BP-5T (EF626692)
82* 59
Pseudogulbenkiania gefcensis yH16T (JF728876)
59*
Crenobacter luteus YIM 78141T (KF771276) Aquaspirillum serpens IAM 13944T (AB074518)
61
Aquaspirillum putridiconchylium IAM 14964T (AB076000) Microvirgula aerodenitrificans SGLY2T (U89333)
87*
Laribacter hongkongensis HLHK9 (CP001154) 100 Laribacter hongkongensis HKU1T (AF389085)
96
Vogesella alkaliphila JC141T (HE819389)
71 84
Vogesella fluminis Npb-07T (JN315669) Vogesella indigofera ATCC 19706T (AB021385) Vogesella lacus GR13T (EU287927)
100* 85*
Vogesella mureinivorans 389T (GQ246809) 99*
Vogesella perlucida DS-28T (EF626691) Neisseria gonorrhoeae NCTC 8375T (X07714)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences (1557 bp) of strain YIM 78141T and the most closely related members of the family Neisseriaceae. Bootstrap values (expressed as percentages of 1000 replications) of above 50 % are shown at branch points. Asterisks denote nodes that were also recovered using the maximum-parsimony and maximum-likelihood methods. Bar, 0.01 substitutions per nucleotide position. Neisseria gonorrhoeae NCTC 8375T (X07714) was used as an outgroup. http://ijs.sgmjournals.org
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(except for cellular fatty acid analysis). Cells of strain YIM 78141T were harvested after 2 days of growth on TSA at 45 uC. Cellular fatty acid analysis was performed using the Sherlock Microbial Identification System (MIDI) according to the manufacturer’s instructions. The major fatty acid components of strain YIM 78141T were C12 : 0 (5.1 %), C16 : 0 (27.2 %), C18 : 1v7c (15.0 %) and summed feature 3 (44.0 %). The major fatty acids of strain YIM 78141T were generally consistent with those of phylogenetically related type strains, Gulbenkiania mobilis E4FC31T, Pseudogulbenkiania subflava BP-5T, Paludibacterium yongneupense 5YN8-15T, Aquitalea magnusonii TRO-001DR8T, Chromobacterium violaceum ATCC 12472T, Chromobacterium subtsugae PRAA4-1T, Vogesella indigofera ATCC 19706T, but there were still many differences in the relative proportions (Table 1). Polar lipids were extracted, examined by two-dimensional TLC and identified using previously described procedures (Minnikin et al., 1979; Collins & Jones, 1980). The polar lipids of strain YIM 78141T consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, phospholipids of unknown structure containing aminoglycophospholipid and three unidentified polar lipids (Fig. S3). Quinones were isolated as described by Collins et al. (1977) and separated by HPLC (Kroppenstedt, 1982). The quinone of strain YIM 78141T was ubiquinone 8. The genomic DNA G+C content was determined by using the HPLC method of Mesbah et al. (1989). The G+C content of the genomic DNA of strain YIM 78141T was 67.3 mol%. Cell morphology was determined with cultures grown for 6, 12, 24, 48 and 72 h on R2A agar at 45 uC. Gram staining was carried out by using the standard Gram reaction and was confirmed by using the KOH lysis test method (Cerny, 1978). Anaerobic growth was evaluated after incubation in an anaerobic chamber for 7 days at 45 uC on an R2A plate. Morphological characteristics of the strain were observed by using light microscopy (model BH 2; Olympus), scanning electron microscopy (XL30 ESEM-TMP; Philips) and transmission electron microscopy (JEM-2100; JEOL). Growth temperature was tested at 4, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 uC on R2A agar plates. For NaCl tolerance experiments, R2A medium was used as the basal medium with NaCl added at concentrations of 0, 1, 2, 3, 4 and 5 % (w/v). The pH range for growth was investigated between pH 4.0 and 11.0 at intervals of 1 pH unit, by using the buffer system described by Xu et al. (2005) (pH 4.0–5.0, 0.1 M citric acid/0.1 M sodium citrate; pH 6.0–8.0, 0.1M KH2PO4/0.1 M NaOH; pH 9.0–10.0, 0.1 M NaHCO3/0.1M Na2CO3; pH 11.0, 0.05M Na2HPO4/0.1M NaOH), and R2A broth as basal medium. Catalase activity was determined, based on the production of bubbles after the addition of a drop of 3 % (w/v) H2O2. Oxidase activity was determined, based on oxidation of tetramethyl-p-phenylenediamine. Hydrolysis of starch (1 %, w/v; Sinopharm Chemical Reagent), casein (1 %, w/v; Sinopharm Chemical Reagent), Tweens 20, 40, 60 and 80 (1 %, w/v; Sigma) and tyrosine (0.5 %, w/v; Solarbio Chemical Reagent) was tested on R2A 216
agar medium. Carbon source assimilation was tested by using Biolog GN3 microplate tests; plates were incubated at 45 uC and read after 48 h. Enzyme activity and other biochemical characteristics were determined by using the API ZYM, API 20 NE and API 50 CH systems (bioMe´rieux) according to the manufacturer’s instructions. Besides the phylogenetic analysis based on 16S rRNA gene sequences, strain YIM 78141T could also be clearly distinguished from other members of the family Neisseriaceae on the basis of DNA G+C content, cellular fatty acid profile and other physical characteristics (Tables 1 and 2). The large difference (about 8.1 %) in the genomic DNA G+C content clearly distinguished strain YIM 78141T from Aquitalea magnusonii TRO-001DR8T (Table 1). Major fatty acids of strain YIM 78141T mainly consisted of C12 : 0, C18 : 1v7c, C16 : 0 and summed feature 3, this being similar to the corresponding data for the closely related taxa (except V. indigofera ATCC 19706T). The major hydroxyl fatty acids of strain YIM 78141T were C10 : 0 3-OH and C12 : 0 3-OH, also similar to the corresponding data for the closely related taxa including G. mobilis E4FC31T, Pseudogulbenkiania subflava BP-5T, Paludibacterium yongneupense 5YN8-15T and Aquitalea magnusonii TRO-001DR8T. However, strain YIM 78141T did not contain C12 : 0 2-OH or C14 : 0, which clearly distinguished it from C. violaceum KCTC 2897T and C. subtsugae PRAA4-1T (Table 1). The results for indole production, activities of arginine dihydrolase and urease, glucose fermentation, and assimilation of maltose,
Table 1. Comparison of cellular fatty acid compositions of strain YIM 78141T and members of phylogenetically related genera Strains: 1, YIM 78141T; 2, G. mobilis E4FC31T; 3, Pseudogulbenkiania subflava BP-5T; 4, Paludibacterium yongneupense 5YN8-15T; 5, Aquitalea magnusonii LMG 23054T; 6, C. violaceum KCTC 2897T; 7, C. subtsugae PRAA4-1T; 8, V. indigofera ATCC 19706T (data from the MIDI database). Data are percentage of total fatty acids; those comprising ,1 % of the total are not listed or are indicated by a dash. Fatty acid C10 : 0 C10 : 0 3-OH C12 : 0 C12 : 0 2-OH C12 : 0 3-OH C14 : 0 C15 : 0 C16 : 0 C17 : 0 cyclo C17 : 1v6c C18 : 1v7c C18 : 0 Summed feature 3*
1 – 2.6 5.1 – 2.3 – – 27.2 – – 15.0 – 44.0
2
3
4
5
2.1 – – – 2.4 2.8 3.0 1.5 3.1 6.0 7.9 5.0 – – – – 2.8 1.9 v3.8 1.2 1.0 – 4.3 2.8 – – – 2.0 28.3 33.6 31.7 21.7 – – 4.5 – – – – 1.2 13.0 9.1 11.2 8.0 1.1 – – – 46.2 43.8 32.1 52.0
6 – 5.2 5.6 3.0 5.0 1.7 – 23.9 1.7 – 15.0 – 35.8
7
8
– – 4.3 5.3 5.0 2.9 2.9 – 4.0 – 3.2 – 1.3 – 25.0 26.6 – – – – 10.6 3.9 – – 41.9 56.9
*Summed feature 3 comprises C16 : 1v7c and/or iso-C15 : 0 2-OH. International Journal of Systematic and Evolutionary Microbiology 65
Crenobacter luteus gen. nov., sp. nov.
Table 2. Differential characteristics among strain YIM 78141T and representatives of related genera Strains: 1, YIM 78141T; 2, G. mobilis E4FC31T; 3, Pseudogulbenkiania subflava BP-5T; 4, Paludibacterium yongneupense 5YN8-15T; 5, Aquitalea magnusonii LMG 23054T; 6, C. violaceum KCTC 2897T; 7, C. subtsugae PRAA4-1T; 7, V. indigofera ATCC 19706T. All data for reference strains are taken from previous studies (Gillis & Logan, 2005; Krieg, 2005; Lau et al., 2006; Martin et al., 2007; Vaz-Moreira et al., 2007; Kwon et al., 2008; Lin et al., 2008). All strains are motile and oxidase-positive. According to API 20NE test strips, all are negative for aesculin hydrolysis, and all assimilate L-histidine, but not L-arabinose, D-mannitol, adipic acid or phenylacetic acid. +, Positive, utilized; 2, negative, not utilized; ND, not determined. Characteristic Colony colour Catalase Growth at: 4 uC 40 uC pH 4 API 20NE Nitrate reduction Indole production Glucose fermentation Arginine dihydrolase Urease Gelatin hydrolysis Assimilation (API 20NE) of: D-Glucose D-Mannose N-Acetylglucosamine Maltose Potassium gluconate Capric acid Malic acid Trisodium citrate Oxidation of (Biolog): Citric acid a-D-Glucose 1-phosphate a-D-Glucose 6-phosphate DL-a-Glycerol phosphate cis-Aconitic acid c-Aminobutyric acid p-Hydroxyphenylacetic acid a-Ketoglutaric acid 2-Aminoethanol 2,3-Butanediol L-Leucine L-Phenylalanine L-Threonine D-Serine L-Alaninamide Inosine Thymidine Uridine DNA G+C content (mol%) Isolation source
1
2
3
4
5
6
7
8
Yellowish +
Cream +
Pale yellow 2
Cream +
Tan +
Violet +
Violet +
Blue +
2 + 2
2 + 2
2 + 2
+ 2 +
2 + 2
2 + 2
+ + 2
+ + 2
+ + + 2 2 2
+ + 2 + + 2
+ 2 2 2 2 ND
+ 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 67.3 Hot spring water
2 2 2 2 2 + 2 + 2 2 + 2 + + + 2 2 2 63.0 Municipal wastewater
2 + + + + 2 + + + + + + + + + + + + 62–64 Cold spring water
ND
+ 2 2 2 + + + + 2 2 + 2 2 2 2 2 2 2 59.2 Humic lake
2 + + + 2 2 2 2 + + 2 + + + + + + + 65–68 Soil and water
2 + + 2 2 2 2 2 2 2 2 2 + + + + 2 2 64.5 Soil
2 2 2 2 2 + 2 + 2 2 + + + 2 + 2 2 2 65.4 Freshwater
potassium gluconate and trisodium citrate were sufficient to distinguish the novel isolate from the closely related strains G. mobilis E4FC31T and Pseudogulbenkiania subflava BP-5T http://ijs.sgmjournals.org
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
63.0 Wetland
(Table 2). The ability not to grow at low temperature or at low pH also distinguished strain YIM 78141T from Paludibacterium yongneupense 5YN8-15T (Table 2). 217
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Therefore, based on the above phenotypic and genotypic differences, we propose that strain YIM 78141T should be considered to represent a novel species within a new genus, for which the name Crenobacter luteus gen. nov., sp. nov. is proposed.
unknown structure containing aminoglycophospholipid and three unidentified polar lipids.
Description of Crenobacter gen. nov.
The type strain YIM 78141T (5BCRC 80650T5KCTC 32558T5DSM 27258T) was isolated from a hot spring sediment sample collected at Hehua hot spring in Tengchong, Yunnan Province, south-west China. The DNA G+C content of the type strain is 67.3 mol%.
Crenobacter (Cre.no.bac9ter. Gr. n. krene a fountain, spring; L. dim. n. bacter small rod; N.L. masc. n. Crenobacter rod from a spring).
Acknowledgements
Cells are slightly thermophilic, Gram-staining-negative, strictly aerobic and short-rod-shaped. Catalase- and oxidase-positive. The major fatty acids are C12 : 0, C18 : 1v7c, C16 : 0 and summed feature 3 (C16 : 1v7c and/or iso-C15 : 0 2OH). The major polar lipids consist of diphosphatidylglycerol, phosphatidylethanolamine and some unknown lipids. Major respiratory quinone is ubiquinone 8. The G+C content of the genomic DNA is approximately 67–68 mol%. Phylogenetically, the genus is affiliated to the family Neisseriaceae. The type species is Crenobacter luteus sp. nov.
The authors are grateful to Dr Deene Manik prabhu (Gulbarga University, India) for his kind comments on this manuscript, and Professor Aharon Oren (The Hebrew University of Jerusalem, Israel) and Dr Elke Lang (DSMZ, Germany) for their comments on Latin names. This research was supported by the Key Project of International Cooperation of China Ministry of Science & Technology (MOST) (no. 2013DFA31980), Key Project of Yunnan Provincial Natural Science Foundation (2013FA004), Research Project of Education Department of Henan Province of China (2011A180025), Henan Provincial Undergraduate Training Programs for Innovation and Entrepreneurship (2011060) and National Science Foundation grant (OISE-0968421). W.-J. L. was also supported by Guangdong Province Higher Vocational Colleges & Schools Pearl River Scholar Funded Scheme (2014).
Description of Crenobacter luteus sp. nov. Crenobacter luteus (lu9te.us. L. adj. luteus yellow). Cells are 0.4–0.6 mm wide and 1.2–1.7 mm long (see Fig. S4). Colonies grown on an R2A agar plates for 2 days are yellowish and circular. Grows at 10–55 uC (optimal growth at 40–50 uC); no growth occurs at 5 uC or 60 uC. The pH range for growth is pH 6.0–10.0 (optimal growth at pH 8.0–9.0). Growth occurs with 0–3 % (w/v) NaCl (optimal growth with 0–1 %) and no growth occurs with 4.0 % (w/v) NaCl. Does not degrade Tween 80, starch or casein. Positive for nitrate reduction, indole production and glucose fermentation; but negative for aesculin hydrolysis, arginine dihydrolase, urease and gelatin hydrolysis. Positive in tests for alkaline phosphatase, C4 esterase, C8 esterase lipase and naphthol-AS-BI-phosphohydrolase; but not for C14 lipase, leucine arylamidase, valine arylamidase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fucosidase, cystine arylamidase, trypsin, chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase or aglucosidase. Acids are produced from arbutin, maltose, glycerol, erythritol, glucose, fructose, ribose, amygdalin, sucrose, D-lyxose, D-tagatose, 5-ketogluconate and mycose. Carbon substrate utilization includes acetic acid, bromosuccinic acid, capric acid, dextrin, a-D-glucose, D-fructose 6-phospate, D-fructose, D-glucose 6-phospate, D-galacturonic acid, D-glucuronic acid, D-malic acid, maltose, trehalose, turanose, D-serine, L-arginine, L-aspartic acid, L-galactonic acid lactone, L-glutamic acid, L-histidine, Llactic acid, L-malic acid, L-serine, glucuronamide, maltose, methyl pyruvate, pectin, propionic acid and sucrose. The only quinone is ubiquinone 8. Major fatty acids (.5 %) are C12 : 0, C18 : 1v7c, C16 : 0 and summed feature 3. The polar lipids consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, phospholipids of 218
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Martin, P. A. W., Gundersen-Rindal, D., Blackburn, M. & Buyer, J. (2007). Chromobacterium subtsugae sp. nov., a betaproteobacterium
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DOI 10.1099/ijs.0.060996-0
Crenobacter luteus gen. nov., sp. nov., isolated from a hot spring Lei Dong,13 Hong Ming,1,23 En-Min Zhou,1 Yi-Rui Yin,1 Lan Liu,1 Hui-Geng Feng,2 Wen-Dong Xian,1 Guo-Xing Nie3 and Wen-Jun Li1,4 Correspondence Wen-Jun Li [email protected] or [email protected]
1
Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education, Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, PR China
2
Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang 453003, PR China
3
College of Fisheries, Henan Normal University, Xinxiang 453007, PR China
4
State Key Laboratory of Biocontrol, Key Laboratory of Biodiversity Dynamics and Conservation of Guangdong Higher Education Institutes, College of Ecology and Evolution, Sun Yat-Sen University, Guangzhou 510275, PR China
A slightly thermophilic, Gram-staining-negative and strictly aerobic bacteria, designated strain YIM 78141T, was isolated from a sediment sample collected at Hehua hot spring, Tengchong, Yunnan province, south-west China. Cells of the strain were short-rod-shaped and colonies were yellowish and circular. The strain grew at pH 6.0–10.0 (optimum, pH 8.0–9.0) and 10–55 6C (optimum, 40–50 6C). Phylogenetic analyses based on 16S rRNA gene sequence comparison demonstrated that strain YIM 78141T belongs to the family Neisseriaceae, and strain YIM 78141T also showed low levels of 16S rRNA gene sequence similarity (below 93.4 %) with all other genera in this family. The only quinone was ubiquinone 8 and the genomic DNA G+C content was 67.3 mol%. Major fatty acids (.5 %) were C12 : 0, C16 : 0, C18 : 1v7c and summed feature 3. The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, phospholipids of unknown structure containing aminoglycophospholipid and three unidentified polar lipids. On the basis of the morphological, physiological and biochemical characteristics as well as genotypic data, this strain should be classified as a representative of a novel genus and species of the family Neisseriaceae, for which the name Crenobacter luteus gen. nov., sp. nov. is proposed. The type strain is YIM 78141T (5BCRC 80650T5KCTC 32558T5 DSM 27258T).
The family Neisseriaceae belongs to the order Neisseriales and comprises 33 genera (http://www.bacterio.net/ -classifgenerafamilies.html#Neisseriaceae). In this family, the major distinguishing features among genera are biochemical characteristics, such as the production of arginine dihydrolase, glucose fermentation and G+C content of the genomic DNA, also supported by low 16S rRNA gene sequence similarities (Grimes et al., 1997; Yuen et al., 2001; Chern et al., 2004; Lau et al., 2006). Members of some of these genera, such as Laribacter and Microvirgula (Yuen et al., 2001; Patureau et al., 1998), do not have the ability to oxidize, ferment or assimilate sugars and have been referred to as asaccharolytic (Yuen et al., 2001). Members 3These authors contributed equally to this work. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain YIM 78141T is KF771276. Four supplementary figures are available with the online Supplementary Material.
214
of these genera have been isolated from various environments, such as soil, water and clinical specimens (Patureau et al., 1998; Yuen et al., 2001; Vela et al., 2005; Lau et al., 2006). During an investigation of thermophilic microbial resources at Hehua hot spring in Tengchong, Yunnan province, southwest China, a slightly thermophilic strain of the family Neisseriaceae, designated YIM 78141T, was isolated from a hot spring sediment sample (24.9728u N 98.39706u E) by using a serial dilution technique. After 1 week of incubation on R2A agar medium (Becton Dickinson, BD) at 45 uC, the colony was picked and restreaked repeatedly onto R2A agar medium at 45 uC, until purity was confirmed. The purified strain was routinely cultivated and stored as aqueous glycerol suspensions (20 %, v/v) at 280 uC. Extraction of genomic DNA and PCR amplification of the 16S rRNA gene sequence were performed as described previously (Cui et al., 2001; Li et al., 2007). The resulting 060996 G 2015 IUMS Printed in Great Britain
Crenobacter luteus gen. nov., sp. nov.
16S rRNA gene sequence was compared with available 16S rRNA gene sequences of cultured species from GenBank via the BLAST program and the EzTaxon-e server (http:// eztaxon-e.ezbiocloud.net/; Kim et al., 2012). Phylogenetic analyses were performed using three tree-making algorithms: neighbour-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981) and maximumparsimony (Fitch, 1971), by using the software package MEGA version 5.0 (Tamura et al., 2011). Kimura’s twoparameter model was used to calculate evolutionary distance matrices of the neighbour-joining method and maximum-likelihood method (Kimura, 1980). The topology of the phylogenetic trees was evaluated by the bootstrap resampling method of Felsenstein (1985) with 1000 resamplings.
by using EzTaxon-e (Kim et al., 2012) indicated that strain YIM 78141T belongs to the family Neisseriaceae of the class Betaproteobacteria; sequence similarities among strain YIM 78141T and the type strains of all other species studied were less than 93.4 %. YIM 78141T was grouped into a larger, compact cluster (59 % bootstrap support) that was composed of the genera Gulbenkiania, Pseudogulbenkiania, Paludibacterium, Aquitalea, Chromobacterium, Aquaspirillum, Microvirgula and Laribacter (Fig. 1). Similar results were obtained using the maximum-parsimony and maximum-likelihood algorithms (Figs S1 and S2, available in the online Supplementary Material). The relatively high sequence divergence values (.6.6 %) showed that the novel isolate was distantly related to the described genera in the family Neisseriaceae.
An almost complete 16S rRNA gene sequence (1557 bp) of strain YIM 78141T was derived, and phylogenetic analysis
Biomass used for chemical studies was obtained from cultures grown on R2A agar medium at 45 uC for 3 days
100* Chromobacterium vaccinii MWU205T (JN120869) Chromobacterium piscinae CCM 3329T (AJ871127) 61
0.01
99
Chromobacterium violaceum ATCC 12472T (AE016825) 100* Chromobacterium pseudoviolaceum CCM 2076T (AJ871128)
93*
Chromobacterium subtsugae PRAA4-1T (AY344056) Chromobacterium aquaticum CC-SEYA-1T (EU109734)
95*
100*
Aquitalea magnusonii TRO-001DR8T (DQ018117)
54
100
Aquitalea denitrificans 5YN1-3T (EU594330) Gulbenkiania indica HT27T (DQ415656)
66* 53*
Chromobacterium haemolyticum MDA0585T (DQ785104)
100 Gulbenkiania mobilis E4FC31T (AM295491) Paludibacterium yongneupense 5YN8-15T (AM396358) Pseudogulbenkiania subflava BP-5T (EF626692)
82* 59
Pseudogulbenkiania gefcensis yH16T (JF728876)
59*
Crenobacter luteus YIM 78141T (KF771276) Aquaspirillum serpens IAM 13944T (AB074518)
61
Aquaspirillum putridiconchylium IAM 14964T (AB076000) Microvirgula aerodenitrificans SGLY2T (U89333)
87*
Laribacter hongkongensis HLHK9 (CP001154) 100 Laribacter hongkongensis HKU1T (AF389085)
96
Vogesella alkaliphila JC141T (HE819389)
71 84
Vogesella fluminis Npb-07T (JN315669) Vogesella indigofera ATCC 19706T (AB021385) Vogesella lacus GR13T (EU287927)
100* 85*
Vogesella mureinivorans 389T (GQ246809) 99*
Vogesella perlucida DS-28T (EF626691) Neisseria gonorrhoeae NCTC 8375T (X07714)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences (1557 bp) of strain YIM 78141T and the most closely related members of the family Neisseriaceae. Bootstrap values (expressed as percentages of 1000 replications) of above 50 % are shown at branch points. Asterisks denote nodes that were also recovered using the maximum-parsimony and maximum-likelihood methods. Bar, 0.01 substitutions per nucleotide position. Neisseria gonorrhoeae NCTC 8375T (X07714) was used as an outgroup. http://ijs.sgmjournals.org
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(except for cellular fatty acid analysis). Cells of strain YIM 78141T were harvested after 2 days of growth on TSA at 45 uC. Cellular fatty acid analysis was performed using the Sherlock Microbial Identification System (MIDI) according to the manufacturer’s instructions. The major fatty acid components of strain YIM 78141T were C12 : 0 (5.1 %), C16 : 0 (27.2 %), C18 : 1v7c (15.0 %) and summed feature 3 (44.0 %). The major fatty acids of strain YIM 78141T were generally consistent with those of phylogenetically related type strains, Gulbenkiania mobilis E4FC31T, Pseudogulbenkiania subflava BP-5T, Paludibacterium yongneupense 5YN8-15T, Aquitalea magnusonii TRO-001DR8T, Chromobacterium violaceum ATCC 12472T, Chromobacterium subtsugae PRAA4-1T, Vogesella indigofera ATCC 19706T, but there were still many differences in the relative proportions (Table 1). Polar lipids were extracted, examined by two-dimensional TLC and identified using previously described procedures (Minnikin et al., 1979; Collins & Jones, 1980). The polar lipids of strain YIM 78141T consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, phospholipids of unknown structure containing aminoglycophospholipid and three unidentified polar lipids (Fig. S3). Quinones were isolated as described by Collins et al. (1977) and separated by HPLC (Kroppenstedt, 1982). The quinone of strain YIM 78141T was ubiquinone 8. The genomic DNA G+C content was determined by using the HPLC method of Mesbah et al. (1989). The G+C content of the genomic DNA of strain YIM 78141T was 67.3 mol%. Cell morphology was determined with cultures grown for 6, 12, 24, 48 and 72 h on R2A agar at 45 uC. Gram staining was carried out by using the standard Gram reaction and was confirmed by using the KOH lysis test method (Cerny, 1978). Anaerobic growth was evaluated after incubation in an anaerobic chamber for 7 days at 45 uC on an R2A plate. Morphological characteristics of the strain were observed by using light microscopy (model BH 2; Olympus), scanning electron microscopy (XL30 ESEM-TMP; Philips) and transmission electron microscopy (JEM-2100; JEOL). Growth temperature was tested at 4, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 uC on R2A agar plates. For NaCl tolerance experiments, R2A medium was used as the basal medium with NaCl added at concentrations of 0, 1, 2, 3, 4 and 5 % (w/v). The pH range for growth was investigated between pH 4.0 and 11.0 at intervals of 1 pH unit, by using the buffer system described by Xu et al. (2005) (pH 4.0–5.0, 0.1 M citric acid/0.1 M sodium citrate; pH 6.0–8.0, 0.1M KH2PO4/0.1 M NaOH; pH 9.0–10.0, 0.1 M NaHCO3/0.1M Na2CO3; pH 11.0, 0.05M Na2HPO4/0.1M NaOH), and R2A broth as basal medium. Catalase activity was determined, based on the production of bubbles after the addition of a drop of 3 % (w/v) H2O2. Oxidase activity was determined, based on oxidation of tetramethyl-p-phenylenediamine. Hydrolysis of starch (1 %, w/v; Sinopharm Chemical Reagent), casein (1 %, w/v; Sinopharm Chemical Reagent), Tweens 20, 40, 60 and 80 (1 %, w/v; Sigma) and tyrosine (0.5 %, w/v; Solarbio Chemical Reagent) was tested on R2A 216
agar medium. Carbon source assimilation was tested by using Biolog GN3 microplate tests; plates were incubated at 45 uC and read after 48 h. Enzyme activity and other biochemical characteristics were determined by using the API ZYM, API 20 NE and API 50 CH systems (bioMe´rieux) according to the manufacturer’s instructions. Besides the phylogenetic analysis based on 16S rRNA gene sequences, strain YIM 78141T could also be clearly distinguished from other members of the family Neisseriaceae on the basis of DNA G+C content, cellular fatty acid profile and other physical characteristics (Tables 1 and 2). The large difference (about 8.1 %) in the genomic DNA G+C content clearly distinguished strain YIM 78141T from Aquitalea magnusonii TRO-001DR8T (Table 1). Major fatty acids of strain YIM 78141T mainly consisted of C12 : 0, C18 : 1v7c, C16 : 0 and summed feature 3, this being similar to the corresponding data for the closely related taxa (except V. indigofera ATCC 19706T). The major hydroxyl fatty acids of strain YIM 78141T were C10 : 0 3-OH and C12 : 0 3-OH, also similar to the corresponding data for the closely related taxa including G. mobilis E4FC31T, Pseudogulbenkiania subflava BP-5T, Paludibacterium yongneupense 5YN8-15T and Aquitalea magnusonii TRO-001DR8T. However, strain YIM 78141T did not contain C12 : 0 2-OH or C14 : 0, which clearly distinguished it from C. violaceum KCTC 2897T and C. subtsugae PRAA4-1T (Table 1). The results for indole production, activities of arginine dihydrolase and urease, glucose fermentation, and assimilation of maltose,
Table 1. Comparison of cellular fatty acid compositions of strain YIM 78141T and members of phylogenetically related genera Strains: 1, YIM 78141T; 2, G. mobilis E4FC31T; 3, Pseudogulbenkiania subflava BP-5T; 4, Paludibacterium yongneupense 5YN8-15T; 5, Aquitalea magnusonii LMG 23054T; 6, C. violaceum KCTC 2897T; 7, C. subtsugae PRAA4-1T; 8, V. indigofera ATCC 19706T (data from the MIDI database). Data are percentage of total fatty acids; those comprising ,1 % of the total are not listed or are indicated by a dash. Fatty acid C10 : 0 C10 : 0 3-OH C12 : 0 C12 : 0 2-OH C12 : 0 3-OH C14 : 0 C15 : 0 C16 : 0 C17 : 0 cyclo C17 : 1v6c C18 : 1v7c C18 : 0 Summed feature 3*
1 – 2.6 5.1 – 2.3 – – 27.2 – – 15.0 – 44.0
2
3
4
5
2.1 – – – 2.4 2.8 3.0 1.5 3.1 6.0 7.9 5.0 – – – – 2.8 1.9 v3.8 1.2 1.0 – 4.3 2.8 – – – 2.0 28.3 33.6 31.7 21.7 – – 4.5 – – – – 1.2 13.0 9.1 11.2 8.0 1.1 – – – 46.2 43.8 32.1 52.0
6 – 5.2 5.6 3.0 5.0 1.7 – 23.9 1.7 – 15.0 – 35.8
7
8
– – 4.3 5.3 5.0 2.9 2.9 – 4.0 – 3.2 – 1.3 – 25.0 26.6 – – – – 10.6 3.9 – – 41.9 56.9
*Summed feature 3 comprises C16 : 1v7c and/or iso-C15 : 0 2-OH. International Journal of Systematic and Evolutionary Microbiology 65
Crenobacter luteus gen. nov., sp. nov.
Table 2. Differential characteristics among strain YIM 78141T and representatives of related genera Strains: 1, YIM 78141T; 2, G. mobilis E4FC31T; 3, Pseudogulbenkiania subflava BP-5T; 4, Paludibacterium yongneupense 5YN8-15T; 5, Aquitalea magnusonii LMG 23054T; 6, C. violaceum KCTC 2897T; 7, C. subtsugae PRAA4-1T; 7, V. indigofera ATCC 19706T. All data for reference strains are taken from previous studies (Gillis & Logan, 2005; Krieg, 2005; Lau et al., 2006; Martin et al., 2007; Vaz-Moreira et al., 2007; Kwon et al., 2008; Lin et al., 2008). All strains are motile and oxidase-positive. According to API 20NE test strips, all are negative for aesculin hydrolysis, and all assimilate L-histidine, but not L-arabinose, D-mannitol, adipic acid or phenylacetic acid. +, Positive, utilized; 2, negative, not utilized; ND, not determined. Characteristic Colony colour Catalase Growth at: 4 uC 40 uC pH 4 API 20NE Nitrate reduction Indole production Glucose fermentation Arginine dihydrolase Urease Gelatin hydrolysis Assimilation (API 20NE) of: D-Glucose D-Mannose N-Acetylglucosamine Maltose Potassium gluconate Capric acid Malic acid Trisodium citrate Oxidation of (Biolog): Citric acid a-D-Glucose 1-phosphate a-D-Glucose 6-phosphate DL-a-Glycerol phosphate cis-Aconitic acid c-Aminobutyric acid p-Hydroxyphenylacetic acid a-Ketoglutaric acid 2-Aminoethanol 2,3-Butanediol L-Leucine L-Phenylalanine L-Threonine D-Serine L-Alaninamide Inosine Thymidine Uridine DNA G+C content (mol%) Isolation source
1
2
3
4
5
6
7
8
Yellowish +
Cream +
Pale yellow 2
Cream +
Tan +
Violet +
Violet +
Blue +
2 + 2
2 + 2
2 + 2
+ 2 +
2 + 2
2 + 2
+ + 2
+ + 2
+ + + 2 2 2
+ + 2 + + 2
+ 2 2 2 2 ND
+ 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 67.3 Hot spring water
2 2 2 2 2 + 2 + 2 2 + 2 + + + 2 2 2 63.0 Municipal wastewater
2 + + + + 2 + + + + + + + + + + + + 62–64 Cold spring water
ND
+ 2 2 2 + + + + 2 2 + 2 2 2 2 2 2 2 59.2 Humic lake
2 + + + 2 2 2 2 + + 2 + + + + + + + 65–68 Soil and water
2 + + 2 2 2 2 2 2 2 2 2 + + + + 2 2 64.5 Soil
2 2 2 2 2 + 2 + 2 2 + + + 2 + 2 2 2 65.4 Freshwater
potassium gluconate and trisodium citrate were sufficient to distinguish the novel isolate from the closely related strains G. mobilis E4FC31T and Pseudogulbenkiania subflava BP-5T http://ijs.sgmjournals.org
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
63.0 Wetland
(Table 2). The ability not to grow at low temperature or at low pH also distinguished strain YIM 78141T from Paludibacterium yongneupense 5YN8-15T (Table 2). 217
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Therefore, based on the above phenotypic and genotypic differences, we propose that strain YIM 78141T should be considered to represent a novel species within a new genus, for which the name Crenobacter luteus gen. nov., sp. nov. is proposed.
unknown structure containing aminoglycophospholipid and three unidentified polar lipids.
Description of Crenobacter gen. nov.
The type strain YIM 78141T (5BCRC 80650T5KCTC 32558T5DSM 27258T) was isolated from a hot spring sediment sample collected at Hehua hot spring in Tengchong, Yunnan Province, south-west China. The DNA G+C content of the type strain is 67.3 mol%.
Crenobacter (Cre.no.bac9ter. Gr. n. krene a fountain, spring; L. dim. n. bacter small rod; N.L. masc. n. Crenobacter rod from a spring).
Acknowledgements
Cells are slightly thermophilic, Gram-staining-negative, strictly aerobic and short-rod-shaped. Catalase- and oxidase-positive. The major fatty acids are C12 : 0, C18 : 1v7c, C16 : 0 and summed feature 3 (C16 : 1v7c and/or iso-C15 : 0 2OH). The major polar lipids consist of diphosphatidylglycerol, phosphatidylethanolamine and some unknown lipids. Major respiratory quinone is ubiquinone 8. The G+C content of the genomic DNA is approximately 67–68 mol%. Phylogenetically, the genus is affiliated to the family Neisseriaceae. The type species is Crenobacter luteus sp. nov.
The authors are grateful to Dr Deene Manik prabhu (Gulbarga University, India) for his kind comments on this manuscript, and Professor Aharon Oren (The Hebrew University of Jerusalem, Israel) and Dr Elke Lang (DSMZ, Germany) for their comments on Latin names. This research was supported by the Key Project of International Cooperation of China Ministry of Science & Technology (MOST) (no. 2013DFA31980), Key Project of Yunnan Provincial Natural Science Foundation (2013FA004), Research Project of Education Department of Henan Province of China (2011A180025), Henan Provincial Undergraduate Training Programs for Innovation and Entrepreneurship (2011060) and National Science Foundation grant (OISE-0968421). W.-J. L. was also supported by Guangdong Province Higher Vocational Colleges & Schools Pearl River Scholar Funded Scheme (2014).
Description of Crenobacter luteus sp. nov. Crenobacter luteus (lu9te.us. L. adj. luteus yellow). Cells are 0.4–0.6 mm wide and 1.2–1.7 mm long (see Fig. S4). Colonies grown on an R2A agar plates for 2 days are yellowish and circular. Grows at 10–55 uC (optimal growth at 40–50 uC); no growth occurs at 5 uC or 60 uC. The pH range for growth is pH 6.0–10.0 (optimal growth at pH 8.0–9.0). Growth occurs with 0–3 % (w/v) NaCl (optimal growth with 0–1 %) and no growth occurs with 4.0 % (w/v) NaCl. Does not degrade Tween 80, starch or casein. Positive for nitrate reduction, indole production and glucose fermentation; but negative for aesculin hydrolysis, arginine dihydrolase, urease and gelatin hydrolysis. Positive in tests for alkaline phosphatase, C4 esterase, C8 esterase lipase and naphthol-AS-BI-phosphohydrolase; but not for C14 lipase, leucine arylamidase, valine arylamidase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fucosidase, cystine arylamidase, trypsin, chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase or aglucosidase. Acids are produced from arbutin, maltose, glycerol, erythritol, glucose, fructose, ribose, amygdalin, sucrose, D-lyxose, D-tagatose, 5-ketogluconate and mycose. Carbon substrate utilization includes acetic acid, bromosuccinic acid, capric acid, dextrin, a-D-glucose, D-fructose 6-phospate, D-fructose, D-glucose 6-phospate, D-galacturonic acid, D-glucuronic acid, D-malic acid, maltose, trehalose, turanose, D-serine, L-arginine, L-aspartic acid, L-galactonic acid lactone, L-glutamic acid, L-histidine, Llactic acid, L-malic acid, L-serine, glucuronamide, maltose, methyl pyruvate, pectin, propionic acid and sucrose. The only quinone is ubiquinone 8. Major fatty acids (.5 %) are C12 : 0, C18 : 1v7c, C16 : 0 and summed feature 3. The polar lipids consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, phospholipids of 218
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