International Journal of Systematic and Evolutionary Microbiology (2014), 64, 3005–3009
DOI 10.1099/ijs.0.061234-0
Chitinimonas prasina sp. nov., isolated from lake water Yi Li,1 Hong Zhu,1 Qiliang Lai,1,2 Xueqian Lei,1 Zhangran Chen,1 Huajun Zhang,1 Yun Tian,1 Wei Zheng1 and Tianling Zheng1 Correspondence Tianling Zheng [email protected] Wei Zheng [email protected]
1
State Key Laboratory for Marine Environmental Sciences and Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystem, School of Life Sciences, Xiamen University, Xiamen 361005, PR China
2
Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, PR China
A Gram-stain-negative, elongated rod-shaped, motile by gliding, green-pigmented, aerobic bacterial strain, designated LY03T, was isolated from lake water in Xiamen, Fujian Province, China. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that the isolate was a member of the genus Chitinimonas, which belongs to the family Burkholderiaceae. Strain LY03T was most closely related to Chitinimonas taiwanensis LMG 22011T (96.02 % 16S rRNA gene sequence similarity), followed by Chitinimonas koreensis KACC 11467T (94.85 %), and the three strains formed a distinct lineage from other strains in the phylogenetic analyses. Optimum conditions for growth were 37 6C, pH 7–9 and without NaCl. The major fatty acids were summed feature 3 (C16 : 1v6c and/or C16 : 1v7c), C16 : 0 and C10 : 0 3-OH. The DNA G+C content of strain LY03T was 63.6 mol% and the major respiratory quinone was ubiquinone-8 (Q-8). The polar lipids were found to consist of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, unknown polar lipids and unidentified phospholipids. Differential phenotypic properties and phylogenetic distinctiveness distinguished strain LY03T from all other members of the genus Chitinimonas. On the basis of its morphology, physiology, fatty acid composition and 16S rRNA gene sequence data, strain LY03T represents a novel species of the genus Chitinimonas, for which the name Chitinimonas prasina sp. nov. is proposed. The type strain is LY03T (5MCCC 1F01209T5KCTC 32574T).
Members of the genus Chitinimonas display the unique ability to degrade chitin. The genus Chitinimonas was first proposed by Chang et al. (2004) and only comprises two species at the time of writing, Chitinimonas taiwanensis and Chitinimonas koreensis (Kim et al., 2006). Comparative 16S rRNA gene sequence analysis indicated that strain LY03T which was isolated from lake water formed a clade within the genus Chitinimonas. Accordingly, the aim of the present work is to determine the exact taxonomic position of strain LY03T by using polyphasic characterization including the determination of phenotypic properties and a detailed phylogenetic analysis based on 16S rRNA gene sequences. A single strain, designated LY03T, was isolated from lake water samples collected in August 2013 at a depth of 0.5 m in Xiamen, Fujian Province, China. Samples were serially The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain LY03T is KF948515. Three supplementary figures are available with the online version of this paper.
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diluted (10-fold dilutions) using sterile distilled water and 0.1 ml aliquots of each dilution were spread onto nutrient agar (NA; Difco) (distilled water 1 l, peptone 5.0 g, meat extract 3.0 g, agar 15.0 g, pH 7.2) followed by incubation for 7 days at 28 uC. Individual colonies of distinct morphology were further purified three times and stored at 280 uC in NA supplemented with 10 % (v/v) glycerol. Genomic DNA was extracted according to the method of Ausubel et al. (1995). The 16S rRNA gene sequence of strain LY03T was amplified by PCR using primers P27F and P1492R (DeLong, 1992). Purification of the PCR product was carried out according to the protocol of the TIANquick midi purification kit (TIANGEN). The purified DNA was cloned into vector pMD19-T and sequenced. Sequences of related taxa were obtained from the GenBank database and the EzTaxon-e database (Kim et al., 2012). Phylogenetic analysis was performed using MEGA software version 4 (Tamura et al., 2007) after multiple alignment of data by DNAMAN (version 5.1; Lynnon Biosoft). Evolutionary distances were determined and clustering was performed by using the neighbour-joining method of 3005
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Saitou & Nei (1987), the maximum-likelihood method and maximum-parsimony method of Tamura et al. (2011) and the minimum evolution method of Rzhetsky & Nei (1993); tree topologies were evaluated by using bootstrap values based on 1000 replications. A nearly full-length 16S rRNA gene sequence (1452 bp) of strain LY03T was determined. Phylogenetic analysis of strain LY03T indicated that it belonged to the genus Chitinimonas (Fig. 1 and Fig. S1 available in the online Supplementary Material). The 16S rRNA gene sequence of strain LY03T showed the highest similarity to Chitinimonas taiwanensis LMG 22011T (96.02 %), and lower similarities were shown to Chitinimonas koreensis KACC 11467T (94.85 %). Strain LY03T formed a separate phylogenetic clade with C. taiwanensis LMG 22011T and C. koreensis KACC 11467T. The values for sequence similarity among the 50 most closely related strains were further determined using the EzTaxon server 2.1 (EzTaxon-e; http://eztaxone.ezbiocloud.net/). The relatively low levels of 16S rRNA gene sequence similarity to the most closely related species indicated that strain LY03T may represent a novel species of the genus Chitinimonas. Cell morphology and motility were observed by using transmission electron microscopy (JEM-2100HC; JEOL) and phase-contrast light microscopy (50i; Nikon), with cells from the early exponential phase grown on NA plates (Fig. S2). Colony morphology, size and colour were examined from cultures on NA plates grown for 24 h. The presence of flexirubin-type pigments was assessed using the bathochromic shift test with 20 % KOH, as described by Bernardet et al. (2002). Gliding motility was investigated as described by Bowman (2000). The Gram reaction was determined by using the bioMe´rieux Gram stain kit according to the manufacturer’s instructions.
98
0.02
90 78
Anaerobic growth was examined according to the protocol of Li et al. (2013a). Growth was tested at 0–42 uC (0, 4, 10, 15, 20, 26, 28, 30, 32, 35, 37, 38, 39, 40 and 42 uC) in NA. The pH range for growth was determined in NA that was adjusted to pH 3.0–12.0 (at 1 pH unit intervals), as previously described (Li et al., 2013b). Verification of the pH after autoclaving revealed only minor changes. The NaCl concentration range and optimum for growth were determined in NA, supplemented with various concentrations of NaCl (0–5.0 % at 0.5 % intervals and 5.0–10.0 % at 1 % intervals; w/v). Catalase activity was determined by addition of 3 % (v/v) hydrogen peroxide to exponentialphase colonies, and the oxidase reaction was tested by using oxidase reagent (bioMe´rieux). Hydrolysis of starch, chitin, gelatin, casein and Tweens 20, 40, 60 and 80, were tested using NA plates, at 0.5 % (w/v) for starch and at 1 % (w/v) for the other substrates. Results were examined twice after growth on agar plates for 2 and 4 days. Further phenotypic and enzymic characterizations of strain LY03T and the reference strains were conducted using the API 20NE and API 50CH test kits (bioMe´rieux) at 28 uC for 2 days and the API ZYM test kit (bioMe´rieux) at 28 uC for 24 h and the API 20E test kit (bioMe´rieux) at 37 uC for 24 h according to the manufacturer’s instructions. All the commercial kits were inoculated with bacterial suspensions in 0.85 % (w/v) NaCl. Susceptibility to antibiotics was tested on NA plates at 37 uC for 24 h by using antibiotic discs (Oxoid) containing the following (mg per disc unless otherwise stated): ampicillin (10), carbenicillin (100), cefalexin (30), cefazolin (30), cefobid (30), cephradin (30), chloramphenicol (30), ciprofloxacin (5), clindamycin (2), cotrimoxazole (25), erythromycin (15), gentamicin (10), kanamycin (30), metronidazole (5), minomycin (30), neomycin (30), norfloxacin (10), novobiocin (5), ofloxacin (5), oxacillin (1), penicillin G (10), piperacillin (100),
Cupriavidus necator N-1T (AF191737) Ralstonia pickettii ATCC 27511T (AY741342) Polynucleobacter necessarius ATCC 30859T (AM397067) Paucimonas lemoignei ATCC 17989T (X92555) Limnobacter thiooxidans CS-K2T (AJ289885)
95
Burkholderia cepacia 717-ICPB 25T (U96927) 99
Pandoraea apista ATCC BAA-61T (AF139173) Chitiniphilus shinanonensis SAY3T (AB453176) Chitinimonas koreensis KACC 11467T (DQ256728)
84 99
Chitinimonas prasina LY03T (KF948515) Chitinimonas taiwanensis LMG 22011T (AY323827) Mycoplana dimorpha TK0055T (D12786)
Fig. 1. Neighbour-joining tree showing the phylogenetic positions of strain LY03T and representatives of some other related taxa, based on 16S rRNA gene sequences. Filled circles indicate nodes that were also recovered in maximum-likelihood, maximum-parsimony and minimum-evolution trees based on the same sequences. Bootstrap values (expressed as percentages of 1000 replications) are shown at branch points; only values .70 % are shown. Mycoplana dimorpha TK0055T (GenBank accession no. D12786) was used as the outgroup. Bar, 0.02 nt substitution rate (Knuc) units. 3006
International Journal of Systematic and Evolutionary Microbiology 64
Chitinimonas prasina sp. nov.
Table 1. Characteristics that differentiate strain LY03T from closely related species of the genus Chitinimonas Strains: 1, LY03T; 2, C. taiwanensis LMG 22011T; 3, C. koreensis KACC 11467T. All strains are Gram-stain-negative rods, display gliding motility, do not produce flexirubin-type pigments and are positive for the hydrolysis of Tweens 20, 40, 60 and 80 and chitin. According to API 20NE strips, all strains are positive for reduction of nitrate, gelatin hydrolysis, utilization of D-glucose and maltose and negative for denitrification, indole production, arginine dihydrolase and urease activities and utilization of L-arabinose, potassium gluconate, capric acid, adipic acid, trisodium citrate and phenylacetic acid. According to the API 20E test, all strains are positive for gelatinase activities and negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, H2S production, urease activities, indole production and acid production from inositol, sorbitol, rhamnose, sucrose, melibiose and amygdalin. According to the API ZYM strip, all strains are positive for alkaline phosphatase, esterase lipase (C8), leucine aminopeptidase, a-chymotrypsin and naphthol-ASBl-phosphoamidase; negative for a-galactosidase, b-glucuronidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and afucosidase. +, Positive; W, weakly positive; 2, negative; ND, not determined. All data were obtained at the same time as part of this study except for growth temperature, salinity range, growth pH and the DNA G+C contents of C. taiwanensis LMG 22011T and C. koreensis KACC 11467T, which are from Chang et al. (2004) and Kim et al. (2006), respectively. Characteristic Colony colour NaCl optimum (%) Temperature for growth (uC) Range Optimum pH for growth Range Optimum Oxidase activity Catalase activity Anaerobic growth Hydrolysis of: Starch Casein Gelatin API ZYM tests Esterase (C4) Valine arylamidase Cystine arylamidase Trypsin Acid phosphatase b-Galactosidase a-Glucosidase API 20NE tests Aesculin hydrolysis D-Mannose D-Mannitol N-acetylglucosamine Malic acid
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1
2
3
Green 0
White 0–1
White 0–1
15–39 37
4–39 25–37
10–40 28–37
4–10 7–9 2 2 2
4–10 6–8 + + +
5–8
2 2 2
+ 2 +
+ + +
+ 2 2
+ + + 2* 2* + 2
+ +D 2 +D +D 2 +D
+ 2 2 2 2
W
2 + + +
2 + 2 2 +
W W
2 W
ND
+ + +
Table 1. cont. Characteristic API 20E tests Citrate utilization Tryptophan deaminase Acetoin production Glucose fermentation Mannitol fermentation Arabinose fermentation Susceptibility to: Carbenicillin Cephradin Cefazolin Penicillin G Piperacillin DNA G+C content (mol%)
1
2
3
2 2 + 2 2 W
2* + 2 + + 2
+ 2 + + 2 2
+ + + + + 63.6
+ + + 2 + 62.8
2 2 2 2 2 65.0
*Results were positive in Chang et al. (2004). DResults were negative in Kim et al. (2006).
polymyxin B (30 U), rifampicin (5), streptomycin (10), sulphafurazole (300), tetracycline (30), trimethoprim (5), vancomycin (30) and vibramycin (30). The physiological and biochemical characteristics of strain LY03T are listed in the species description and in Table 1. For cellular fatty acid analysis, fatty acids of strain LY03T, C. taiwanensis LMG 22011T and C. koreensis KACC 11467T cells grown on NA plates at 37 uC for 24 h were extracted, saponified and esterified at the same time according to the standard protocol of the Microbial Identification System (Sasser, 1990). The three strains had similar growth rates at 37 uC and had same physiological age after growth on NA plates at 37 uC for 24 h. The fatty acids were analysed using a gas chromatograph (6850; Agilent), and peaks were identified with the MIDI software (version 6.0). As shown in Table 2, the major fatty acids of strain LY03T were summed feature 3 (C16 : 1v6c and/or C16 : 1v7c) (41.61 %), C16 : 0 (26.89 %) and C10 : 0 3-OH (9.20 %), which accounted for 77.7 %. Compared with C. taiwanensis LMG 22011T, strain LY03T did not possess C12 : 0 2-OH and possessed a lower content of C12 : 0 (2.75 %), C. taiwanensis LMG 22011T possessed C12 : 0 2-OH and a higher content of C12 : 0 (12.41 %). There were also great distinctions between strain LY03T and C. koreensis KACC 11467T. To determine the DNA G+C content, genomic DNA was extracted from cells that had been cultured on NA plates for 24 h at 37 uC and analysed by reverse-phase HPLC (Tamaoka & Komagata, 1984). The DNA G+C content of strain LY03T was 63.6 mol%, which was similar to the two reference strains of species of the genus Chitinimonas. The major respiratory quinone of strain LY03T was determined to be ubiquinone-8 (Q-8) which was determined by the Identification Service of the Leibniz-Institut Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, 3007
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Table 2. Cellular fatty acid contents of strain LY03T and closely related members of the genus Chitinimonas Strains: 1, LY03T; 2, C. taiwanensis LMG 22011T; 3, C. koreensis KACC 11467T. All data are from this study. Values are percentages of total fatty acids. TR, Trace (,0.5 %); ND, not detected. Fatty acid C10 : 0 C10 : 0 3-OH C12 : 0 C12 : 0 2-OH C14 : 0 C14 : 1v5c C15 : 1v5c C16 : 0 C16 : 0 Nalcohol C16 : 1v7calcohol C17 : 0 10methyl anteisoC17 : 1v9c C18 : 0 Summed feature* 3 8
1
2
3.94 9.20 2.75
0.80 13.90 12.41 5.00 1.69
ND
4.29 2.47 0.84 26.89 0.54
TR
3 TR
8.63 9.30 6.94 3.30 0.52
TR
TR
20.94 0.68
19.48 6.00
TR
1.66
5.19
ND
TR
0.52
ND
ND
1.91
TR
0.70
0.67
33.92 4.33
29.99 2.08
LY03T formed smooth, circular, shiny, green colonies and cells had a short flagellum and were capsular, which differed from the white colonies, long flagellum and noncapsular C. taiwanensis LMG 22011T and also from the sticky, white colonies of C. koreensis KACC 11467T. There were also differences in oxidase and catalase activities, anaerobic growth and the degradation of starch, casein and gelatin as well as acid production from glucose, mannitol and arabinose. Strain LY03T was susceptible to carbenicillin, cephradin, cefazolin, penicillin G and piperacillin whilst C. koreensis KACC 11467T was resistant. The dominant fatty acids of strain LY03T and the two reference strains showed huge differences. On the basis of morphological, physiological and chemotaxonomic characteristics, together with data from 16S rRNA gene sequences comparison described above, strain LY03T should be classified as representing a novel species within the genus Chitinimonas, for which the name Chitinimonas prasina sp. nov. is proposed. Description of Chitinimonas prasina sp. nov. Chitinimonas prasina (pra9si.na L. fem. adj. prasina green).
Comparisons of strain LY03T with the two reference strains revealed many phenotypic differences. On NA plates, strain
Cells are Gram-stain-negative, rod-shaped (0.6–0.8 mm in diameter and 2.2–4.2 mm in length) capsular with a flagellum (Fig. S2) and motile by gliding. Growth is visible after 24 h of incubation on NA plates at 37 uC. Colonies on NA plates are shiny, green and circular with regular, smooth edges and are 1–2 mm in diameter after incubation for 48 h at 37 uC. Growth occurs at 15–39 uC, with an optimum at 37 uC. Growth occurs in the presence of 0–1 % (w/v) NaCl; optimal growth occurs without NaCl. Growth occurs at pH 4–10, with optimal growth at pH 7–9. Growth does not occur under anaerobic conditions on NA. Flexirubin-type pigments are not produced. Positive for reduction of nitrate and hydrolysis of chitin and Tweens 20, 40, 60 and 80. Negative for oxidase and catalase activities, denitrification and hydrolysis of starch, gelatin and casein and production of H2S. According to API 20NE strips, positive for b-glucosidase (aesculin hydrolysis), gelatin hydrolysis, utilization of D-glucose and maltose; negative for denitrification, indole production, arginine dihydrolase, urease activity, b-galactosidase and utilization of L-arabinose, D-mannose, D-mannitol, N-acetylglucosamine, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. According to API 20E strips, positive for acetoin production and gelatinase and acid production from arabinose; negative for b-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate utilization, H2S production, urease activities, tryptophan deaminase, indole production and acid production from glucose, mannitol, inositol, sorbitol, rhamnose, melibiose, sucrose and amygdalin. In tests using the API 50CH kit, acid is produced from D-glucose, aesculin-ferric-citrate, maltose, starch, glycogen and potassium 5-ketogluconate. According to the API ZYM kit, positive for alkaline phosphatase, esterase lipase (C8),
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International Journal of Systematic and Evolutionary Microbiology 64
41.61 4.84
*Summed features represent groups of two or more fatty acids that could not be separated by GLC with the MIDI system. Summed feature 3 contained C16 : 1v6c and/or C16 : 1v7c; summed feature 8 contained C18 : 1v7c and/or C18 : 1v6c.
Braunschweig, Germany). This result was in accordance with the properties of the members of the genus Chitinimonas. Polar lipids of strain LY03T, C. taiwanensis LMG 22011T and C. koreensis KACC 11467T (Fig. S3) were extracted using a chloroform/methanol system and analysed using one- and two-dimensional TLC, as described previously (Kates, 1986). Merck silica gel 60 F254 aluminium-backed thin-layer plates were used in the TLC analyses. The plate dotted with sample was subjected to two-dimensional development, with the first solvent of chloroform/methanol/water (65 : 25 : 4, by vol.) followed by second solvent of chloroform/methanol/acetic acid/water (85 : 12 : 15 : 4, by vol.). The main polar lipids of strain LY03T were found to consist of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, unknown polar lipids and unidentified phospholipids. Compared with the closed two strains, unidentified phospholipid (PL1–PL4) were found in strain LY03T, only PL1 was found in C. taiwanensis LMG 22011T, and there were no unidentified phospholipids in C. koreensis KACC 11467T.
Chitinimonas prasina sp. nov.
leucine aminopeptidase, a-chymotrypsin, acid phosphatase and naphthol-AS-Bl-phosphoamidase; weakly positive for esterase (C4), lipase (C14), valine arylamidase and trypsin; negative for cystine arylamidase, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, Nacetyl-b-glucosaminidase, a-mannosidase and a-fucosidase. Sensitive to carbenicillin, cefalexin, cefazolin, cephradin, chloramphenicol, ciprofloxacin, cotrimoxazole, erythromycin, gentamicin, kanamycin, minomycin, neomycin, norfloxacin, novobiocin, ofloxacin, penicillin G, piperacillin, polymyxin B, rifampicin, streptomycin, sulphafurazole, tetracycline, trimethoprim, vancomycin and vibramycin; resistant to ampicillin, cefobid, clindamycin, metronidazole and oxacillin. The predominant fatty acids are summed feature 3 (C16 : 1v6c and/or C16 : 1v7c), C16 : 0 and C10 : 0 3OH. The predominant respiratory quinone is ubiquinone-8 (Q-8). The main polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, unknown polar lipids and unidentified phospholipids. Other phenotypic characteristics are given in Table 1. The type strain, LY03T (5MCCC 1F01209T5KCTC 32574T) was isolated from lake water in Xiamen, Fujian Province, China. The DNA G+C content of the type strain is 63.6 mol%.
Bowman, J. P. (2000). Description of Cellulophaga algicola sp. nov.,
isolated from the surfaces of Antarctic algae, and reclassification of Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Cellulophaga uliginosa comb. nov. Int J Syst Evol Microbiol 50, 1861– 1868. Chang, S.-C., Wang, J.-T., Vandamme, P., Hwang, J.-H., Chang, P.-S. & Chen, W.-M. (2004). Chitinimonas taiwanensis gen. nov., sp. nov., a
novel chitinolytic bacterium isolated from a freshwater pond for shrimp culture. Syst Appl Microbiol 27, 43–49. DeLong, E. F. (1992). Archaea in coastal marine environments. Proc
Natl Acad Sci U S A 89, 5685–5689. Kates, M. (1986). Techniques of Lipidology isolation, analysis and identification of lipids, 2nd ed. rev., pp. 106–107, 241–246. Amsterdam: Elsevier. Kim, B.-Y., Weon, H.-Y., Yoo, S.-H., Chen, W.-M., Kwon, S.-W., Go, S.-J. & Stackebrandt, E. (2006). Chitinimonas koreensis sp. nov.,
isolated from greenhouse soil in Korea. Int J Syst Evol Microbiol 56, 1761–1764. Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Jeon, Y. S., Lee, J. H. & other authors (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. Li, Y., Bai, S., Yang, C., Lai, Q., Zhang, H., Chen, Z., Wei, J., Zheng, W., Tian, Y. & Zheng, T. (2013a). Mangrovimonas yunxiaonensis gen. nov.,
sp. nov., isolated from mangrove sediment. Int J Syst Evol Microbiol 63, 2043–2048. Li, Y., Wei, J., Yang, C., Lai, Q., Chen, Z., Li, D., Zhang, H., Tian, Y., Zheng, W. & Zheng, T. (2013b). Tenacibaculum xiamenense sp. nov.,
Acknowledgements This work was supported by the Public Science and Technology Research Funds Projects of Ocean (201305016, 201305022), National Nature Science Foundation of China (40930847, 41376119) and Science and Technology Innovation Funds of Shenzhen (JCYJ20120615161239998). We would like to thank Professor I. J. Hodgkiss from Hong Kong University for help with English.
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DOI 10.1099/ijs.0.061234-0
Chitinimonas prasina sp. nov., isolated from lake water Yi Li,1 Hong Zhu,1 Qiliang Lai,1,2 Xueqian Lei,1 Zhangran Chen,1 Huajun Zhang,1 Yun Tian,1 Wei Zheng1 and Tianling Zheng1 Correspondence Tianling Zheng [email protected] Wei Zheng [email protected]
1
State Key Laboratory for Marine Environmental Sciences and Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystem, School of Life Sciences, Xiamen University, Xiamen 361005, PR China
2
Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, PR China
A Gram-stain-negative, elongated rod-shaped, motile by gliding, green-pigmented, aerobic bacterial strain, designated LY03T, was isolated from lake water in Xiamen, Fujian Province, China. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that the isolate was a member of the genus Chitinimonas, which belongs to the family Burkholderiaceae. Strain LY03T was most closely related to Chitinimonas taiwanensis LMG 22011T (96.02 % 16S rRNA gene sequence similarity), followed by Chitinimonas koreensis KACC 11467T (94.85 %), and the three strains formed a distinct lineage from other strains in the phylogenetic analyses. Optimum conditions for growth were 37 6C, pH 7–9 and without NaCl. The major fatty acids were summed feature 3 (C16 : 1v6c and/or C16 : 1v7c), C16 : 0 and C10 : 0 3-OH. The DNA G+C content of strain LY03T was 63.6 mol% and the major respiratory quinone was ubiquinone-8 (Q-8). The polar lipids were found to consist of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, unknown polar lipids and unidentified phospholipids. Differential phenotypic properties and phylogenetic distinctiveness distinguished strain LY03T from all other members of the genus Chitinimonas. On the basis of its morphology, physiology, fatty acid composition and 16S rRNA gene sequence data, strain LY03T represents a novel species of the genus Chitinimonas, for which the name Chitinimonas prasina sp. nov. is proposed. The type strain is LY03T (5MCCC 1F01209T5KCTC 32574T).
Members of the genus Chitinimonas display the unique ability to degrade chitin. The genus Chitinimonas was first proposed by Chang et al. (2004) and only comprises two species at the time of writing, Chitinimonas taiwanensis and Chitinimonas koreensis (Kim et al., 2006). Comparative 16S rRNA gene sequence analysis indicated that strain LY03T which was isolated from lake water formed a clade within the genus Chitinimonas. Accordingly, the aim of the present work is to determine the exact taxonomic position of strain LY03T by using polyphasic characterization including the determination of phenotypic properties and a detailed phylogenetic analysis based on 16S rRNA gene sequences. A single strain, designated LY03T, was isolated from lake water samples collected in August 2013 at a depth of 0.5 m in Xiamen, Fujian Province, China. Samples were serially The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain LY03T is KF948515. Three supplementary figures are available with the online version of this paper.
061234 G 2014 IUMS
Printed in Great Britain
diluted (10-fold dilutions) using sterile distilled water and 0.1 ml aliquots of each dilution were spread onto nutrient agar (NA; Difco) (distilled water 1 l, peptone 5.0 g, meat extract 3.0 g, agar 15.0 g, pH 7.2) followed by incubation for 7 days at 28 uC. Individual colonies of distinct morphology were further purified three times and stored at 280 uC in NA supplemented with 10 % (v/v) glycerol. Genomic DNA was extracted according to the method of Ausubel et al. (1995). The 16S rRNA gene sequence of strain LY03T was amplified by PCR using primers P27F and P1492R (DeLong, 1992). Purification of the PCR product was carried out according to the protocol of the TIANquick midi purification kit (TIANGEN). The purified DNA was cloned into vector pMD19-T and sequenced. Sequences of related taxa were obtained from the GenBank database and the EzTaxon-e database (Kim et al., 2012). Phylogenetic analysis was performed using MEGA software version 4 (Tamura et al., 2007) after multiple alignment of data by DNAMAN (version 5.1; Lynnon Biosoft). Evolutionary distances were determined and clustering was performed by using the neighbour-joining method of 3005
Y. Li and others
Saitou & Nei (1987), the maximum-likelihood method and maximum-parsimony method of Tamura et al. (2011) and the minimum evolution method of Rzhetsky & Nei (1993); tree topologies were evaluated by using bootstrap values based on 1000 replications. A nearly full-length 16S rRNA gene sequence (1452 bp) of strain LY03T was determined. Phylogenetic analysis of strain LY03T indicated that it belonged to the genus Chitinimonas (Fig. 1 and Fig. S1 available in the online Supplementary Material). The 16S rRNA gene sequence of strain LY03T showed the highest similarity to Chitinimonas taiwanensis LMG 22011T (96.02 %), and lower similarities were shown to Chitinimonas koreensis KACC 11467T (94.85 %). Strain LY03T formed a separate phylogenetic clade with C. taiwanensis LMG 22011T and C. koreensis KACC 11467T. The values for sequence similarity among the 50 most closely related strains were further determined using the EzTaxon server 2.1 (EzTaxon-e; http://eztaxone.ezbiocloud.net/). The relatively low levels of 16S rRNA gene sequence similarity to the most closely related species indicated that strain LY03T may represent a novel species of the genus Chitinimonas. Cell morphology and motility were observed by using transmission electron microscopy (JEM-2100HC; JEOL) and phase-contrast light microscopy (50i; Nikon), with cells from the early exponential phase grown on NA plates (Fig. S2). Colony morphology, size and colour were examined from cultures on NA plates grown for 24 h. The presence of flexirubin-type pigments was assessed using the bathochromic shift test with 20 % KOH, as described by Bernardet et al. (2002). Gliding motility was investigated as described by Bowman (2000). The Gram reaction was determined by using the bioMe´rieux Gram stain kit according to the manufacturer’s instructions.
98
0.02
90 78
Anaerobic growth was examined according to the protocol of Li et al. (2013a). Growth was tested at 0–42 uC (0, 4, 10, 15, 20, 26, 28, 30, 32, 35, 37, 38, 39, 40 and 42 uC) in NA. The pH range for growth was determined in NA that was adjusted to pH 3.0–12.0 (at 1 pH unit intervals), as previously described (Li et al., 2013b). Verification of the pH after autoclaving revealed only minor changes. The NaCl concentration range and optimum for growth were determined in NA, supplemented with various concentrations of NaCl (0–5.0 % at 0.5 % intervals and 5.0–10.0 % at 1 % intervals; w/v). Catalase activity was determined by addition of 3 % (v/v) hydrogen peroxide to exponentialphase colonies, and the oxidase reaction was tested by using oxidase reagent (bioMe´rieux). Hydrolysis of starch, chitin, gelatin, casein and Tweens 20, 40, 60 and 80, were tested using NA plates, at 0.5 % (w/v) for starch and at 1 % (w/v) for the other substrates. Results were examined twice after growth on agar plates for 2 and 4 days. Further phenotypic and enzymic characterizations of strain LY03T and the reference strains were conducted using the API 20NE and API 50CH test kits (bioMe´rieux) at 28 uC for 2 days and the API ZYM test kit (bioMe´rieux) at 28 uC for 24 h and the API 20E test kit (bioMe´rieux) at 37 uC for 24 h according to the manufacturer’s instructions. All the commercial kits were inoculated with bacterial suspensions in 0.85 % (w/v) NaCl. Susceptibility to antibiotics was tested on NA plates at 37 uC for 24 h by using antibiotic discs (Oxoid) containing the following (mg per disc unless otherwise stated): ampicillin (10), carbenicillin (100), cefalexin (30), cefazolin (30), cefobid (30), cephradin (30), chloramphenicol (30), ciprofloxacin (5), clindamycin (2), cotrimoxazole (25), erythromycin (15), gentamicin (10), kanamycin (30), metronidazole (5), minomycin (30), neomycin (30), norfloxacin (10), novobiocin (5), ofloxacin (5), oxacillin (1), penicillin G (10), piperacillin (100),
Cupriavidus necator N-1T (AF191737) Ralstonia pickettii ATCC 27511T (AY741342) Polynucleobacter necessarius ATCC 30859T (AM397067) Paucimonas lemoignei ATCC 17989T (X92555) Limnobacter thiooxidans CS-K2T (AJ289885)
95
Burkholderia cepacia 717-ICPB 25T (U96927) 99
Pandoraea apista ATCC BAA-61T (AF139173) Chitiniphilus shinanonensis SAY3T (AB453176) Chitinimonas koreensis KACC 11467T (DQ256728)
84 99
Chitinimonas prasina LY03T (KF948515) Chitinimonas taiwanensis LMG 22011T (AY323827) Mycoplana dimorpha TK0055T (D12786)
Fig. 1. Neighbour-joining tree showing the phylogenetic positions of strain LY03T and representatives of some other related taxa, based on 16S rRNA gene sequences. Filled circles indicate nodes that were also recovered in maximum-likelihood, maximum-parsimony and minimum-evolution trees based on the same sequences. Bootstrap values (expressed as percentages of 1000 replications) are shown at branch points; only values .70 % are shown. Mycoplana dimorpha TK0055T (GenBank accession no. D12786) was used as the outgroup. Bar, 0.02 nt substitution rate (Knuc) units. 3006
International Journal of Systematic and Evolutionary Microbiology 64
Chitinimonas prasina sp. nov.
Table 1. Characteristics that differentiate strain LY03T from closely related species of the genus Chitinimonas Strains: 1, LY03T; 2, C. taiwanensis LMG 22011T; 3, C. koreensis KACC 11467T. All strains are Gram-stain-negative rods, display gliding motility, do not produce flexirubin-type pigments and are positive for the hydrolysis of Tweens 20, 40, 60 and 80 and chitin. According to API 20NE strips, all strains are positive for reduction of nitrate, gelatin hydrolysis, utilization of D-glucose and maltose and negative for denitrification, indole production, arginine dihydrolase and urease activities and utilization of L-arabinose, potassium gluconate, capric acid, adipic acid, trisodium citrate and phenylacetic acid. According to the API 20E test, all strains are positive for gelatinase activities and negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, H2S production, urease activities, indole production and acid production from inositol, sorbitol, rhamnose, sucrose, melibiose and amygdalin. According to the API ZYM strip, all strains are positive for alkaline phosphatase, esterase lipase (C8), leucine aminopeptidase, a-chymotrypsin and naphthol-ASBl-phosphoamidase; negative for a-galactosidase, b-glucuronidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and afucosidase. +, Positive; W, weakly positive; 2, negative; ND, not determined. All data were obtained at the same time as part of this study except for growth temperature, salinity range, growth pH and the DNA G+C contents of C. taiwanensis LMG 22011T and C. koreensis KACC 11467T, which are from Chang et al. (2004) and Kim et al. (2006), respectively. Characteristic Colony colour NaCl optimum (%) Temperature for growth (uC) Range Optimum pH for growth Range Optimum Oxidase activity Catalase activity Anaerobic growth Hydrolysis of: Starch Casein Gelatin API ZYM tests Esterase (C4) Valine arylamidase Cystine arylamidase Trypsin Acid phosphatase b-Galactosidase a-Glucosidase API 20NE tests Aesculin hydrolysis D-Mannose D-Mannitol N-acetylglucosamine Malic acid
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1
2
3
Green 0
White 0–1
White 0–1
15–39 37
4–39 25–37
10–40 28–37
4–10 7–9 2 2 2
4–10 6–8 + + +
5–8
2 2 2
+ 2 +
+ + +
+ 2 2
+ + + 2* 2* + 2
+ +D 2 +D +D 2 +D
+ 2 2 2 2
W
2 + + +
2 + 2 2 +
W W
2 W
ND
+ + +
Table 1. cont. Characteristic API 20E tests Citrate utilization Tryptophan deaminase Acetoin production Glucose fermentation Mannitol fermentation Arabinose fermentation Susceptibility to: Carbenicillin Cephradin Cefazolin Penicillin G Piperacillin DNA G+C content (mol%)
1
2
3
2 2 + 2 2 W
2* + 2 + + 2
+ 2 + + 2 2
+ + + + + 63.6
+ + + 2 + 62.8
2 2 2 2 2 65.0
*Results were positive in Chang et al. (2004). DResults were negative in Kim et al. (2006).
polymyxin B (30 U), rifampicin (5), streptomycin (10), sulphafurazole (300), tetracycline (30), trimethoprim (5), vancomycin (30) and vibramycin (30). The physiological and biochemical characteristics of strain LY03T are listed in the species description and in Table 1. For cellular fatty acid analysis, fatty acids of strain LY03T, C. taiwanensis LMG 22011T and C. koreensis KACC 11467T cells grown on NA plates at 37 uC for 24 h were extracted, saponified and esterified at the same time according to the standard protocol of the Microbial Identification System (Sasser, 1990). The three strains had similar growth rates at 37 uC and had same physiological age after growth on NA plates at 37 uC for 24 h. The fatty acids were analysed using a gas chromatograph (6850; Agilent), and peaks were identified with the MIDI software (version 6.0). As shown in Table 2, the major fatty acids of strain LY03T were summed feature 3 (C16 : 1v6c and/or C16 : 1v7c) (41.61 %), C16 : 0 (26.89 %) and C10 : 0 3-OH (9.20 %), which accounted for 77.7 %. Compared with C. taiwanensis LMG 22011T, strain LY03T did not possess C12 : 0 2-OH and possessed a lower content of C12 : 0 (2.75 %), C. taiwanensis LMG 22011T possessed C12 : 0 2-OH and a higher content of C12 : 0 (12.41 %). There were also great distinctions between strain LY03T and C. koreensis KACC 11467T. To determine the DNA G+C content, genomic DNA was extracted from cells that had been cultured on NA plates for 24 h at 37 uC and analysed by reverse-phase HPLC (Tamaoka & Komagata, 1984). The DNA G+C content of strain LY03T was 63.6 mol%, which was similar to the two reference strains of species of the genus Chitinimonas. The major respiratory quinone of strain LY03T was determined to be ubiquinone-8 (Q-8) which was determined by the Identification Service of the Leibniz-Institut Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, 3007
Y. Li and others
Table 2. Cellular fatty acid contents of strain LY03T and closely related members of the genus Chitinimonas Strains: 1, LY03T; 2, C. taiwanensis LMG 22011T; 3, C. koreensis KACC 11467T. All data are from this study. Values are percentages of total fatty acids. TR, Trace (,0.5 %); ND, not detected. Fatty acid C10 : 0 C10 : 0 3-OH C12 : 0 C12 : 0 2-OH C14 : 0 C14 : 1v5c C15 : 1v5c C16 : 0 C16 : 0 Nalcohol C16 : 1v7calcohol C17 : 0 10methyl anteisoC17 : 1v9c C18 : 0 Summed feature* 3 8
1
2
3.94 9.20 2.75
0.80 13.90 12.41 5.00 1.69
ND
4.29 2.47 0.84 26.89 0.54
TR
3 TR
8.63 9.30 6.94 3.30 0.52
TR
TR
20.94 0.68
19.48 6.00
TR
1.66
5.19
ND
TR
0.52
ND
ND
1.91
TR
0.70
0.67
33.92 4.33
29.99 2.08
LY03T formed smooth, circular, shiny, green colonies and cells had a short flagellum and were capsular, which differed from the white colonies, long flagellum and noncapsular C. taiwanensis LMG 22011T and also from the sticky, white colonies of C. koreensis KACC 11467T. There were also differences in oxidase and catalase activities, anaerobic growth and the degradation of starch, casein and gelatin as well as acid production from glucose, mannitol and arabinose. Strain LY03T was susceptible to carbenicillin, cephradin, cefazolin, penicillin G and piperacillin whilst C. koreensis KACC 11467T was resistant. The dominant fatty acids of strain LY03T and the two reference strains showed huge differences. On the basis of morphological, physiological and chemotaxonomic characteristics, together with data from 16S rRNA gene sequences comparison described above, strain LY03T should be classified as representing a novel species within the genus Chitinimonas, for which the name Chitinimonas prasina sp. nov. is proposed. Description of Chitinimonas prasina sp. nov. Chitinimonas prasina (pra9si.na L. fem. adj. prasina green).
Comparisons of strain LY03T with the two reference strains revealed many phenotypic differences. On NA plates, strain
Cells are Gram-stain-negative, rod-shaped (0.6–0.8 mm in diameter and 2.2–4.2 mm in length) capsular with a flagellum (Fig. S2) and motile by gliding. Growth is visible after 24 h of incubation on NA plates at 37 uC. Colonies on NA plates are shiny, green and circular with regular, smooth edges and are 1–2 mm in diameter after incubation for 48 h at 37 uC. Growth occurs at 15–39 uC, with an optimum at 37 uC. Growth occurs in the presence of 0–1 % (w/v) NaCl; optimal growth occurs without NaCl. Growth occurs at pH 4–10, with optimal growth at pH 7–9. Growth does not occur under anaerobic conditions on NA. Flexirubin-type pigments are not produced. Positive for reduction of nitrate and hydrolysis of chitin and Tweens 20, 40, 60 and 80. Negative for oxidase and catalase activities, denitrification and hydrolysis of starch, gelatin and casein and production of H2S. According to API 20NE strips, positive for b-glucosidase (aesculin hydrolysis), gelatin hydrolysis, utilization of D-glucose and maltose; negative for denitrification, indole production, arginine dihydrolase, urease activity, b-galactosidase and utilization of L-arabinose, D-mannose, D-mannitol, N-acetylglucosamine, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. According to API 20E strips, positive for acetoin production and gelatinase and acid production from arabinose; negative for b-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate utilization, H2S production, urease activities, tryptophan deaminase, indole production and acid production from glucose, mannitol, inositol, sorbitol, rhamnose, melibiose, sucrose and amygdalin. In tests using the API 50CH kit, acid is produced from D-glucose, aesculin-ferric-citrate, maltose, starch, glycogen and potassium 5-ketogluconate. According to the API ZYM kit, positive for alkaline phosphatase, esterase lipase (C8),
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International Journal of Systematic and Evolutionary Microbiology 64
41.61 4.84
*Summed features represent groups of two or more fatty acids that could not be separated by GLC with the MIDI system. Summed feature 3 contained C16 : 1v6c and/or C16 : 1v7c; summed feature 8 contained C18 : 1v7c and/or C18 : 1v6c.
Braunschweig, Germany). This result was in accordance with the properties of the members of the genus Chitinimonas. Polar lipids of strain LY03T, C. taiwanensis LMG 22011T and C. koreensis KACC 11467T (Fig. S3) were extracted using a chloroform/methanol system and analysed using one- and two-dimensional TLC, as described previously (Kates, 1986). Merck silica gel 60 F254 aluminium-backed thin-layer plates were used in the TLC analyses. The plate dotted with sample was subjected to two-dimensional development, with the first solvent of chloroform/methanol/water (65 : 25 : 4, by vol.) followed by second solvent of chloroform/methanol/acetic acid/water (85 : 12 : 15 : 4, by vol.). The main polar lipids of strain LY03T were found to consist of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, unknown polar lipids and unidentified phospholipids. Compared with the closed two strains, unidentified phospholipid (PL1–PL4) were found in strain LY03T, only PL1 was found in C. taiwanensis LMG 22011T, and there were no unidentified phospholipids in C. koreensis KACC 11467T.
Chitinimonas prasina sp. nov.
leucine aminopeptidase, a-chymotrypsin, acid phosphatase and naphthol-AS-Bl-phosphoamidase; weakly positive for esterase (C4), lipase (C14), valine arylamidase and trypsin; negative for cystine arylamidase, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, Nacetyl-b-glucosaminidase, a-mannosidase and a-fucosidase. Sensitive to carbenicillin, cefalexin, cefazolin, cephradin, chloramphenicol, ciprofloxacin, cotrimoxazole, erythromycin, gentamicin, kanamycin, minomycin, neomycin, norfloxacin, novobiocin, ofloxacin, penicillin G, piperacillin, polymyxin B, rifampicin, streptomycin, sulphafurazole, tetracycline, trimethoprim, vancomycin and vibramycin; resistant to ampicillin, cefobid, clindamycin, metronidazole and oxacillin. The predominant fatty acids are summed feature 3 (C16 : 1v6c and/or C16 : 1v7c), C16 : 0 and C10 : 0 3OH. The predominant respiratory quinone is ubiquinone-8 (Q-8). The main polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, unknown polar lipids and unidentified phospholipids. Other phenotypic characteristics are given in Table 1. The type strain, LY03T (5MCCC 1F01209T5KCTC 32574T) was isolated from lake water in Xiamen, Fujian Province, China. The DNA G+C content of the type strain is 63.6 mol%.
Bowman, J. P. (2000). Description of Cellulophaga algicola sp. nov.,
isolated from the surfaces of Antarctic algae, and reclassification of Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Cellulophaga uliginosa comb. nov. Int J Syst Evol Microbiol 50, 1861– 1868. Chang, S.-C., Wang, J.-T., Vandamme, P., Hwang, J.-H., Chang, P.-S. & Chen, W.-M. (2004). Chitinimonas taiwanensis gen. nov., sp. nov., a
novel chitinolytic bacterium isolated from a freshwater pond for shrimp culture. Syst Appl Microbiol 27, 43–49. DeLong, E. F. (1992). Archaea in coastal marine environments. Proc
Natl Acad Sci U S A 89, 5685–5689. Kates, M. (1986). Techniques of Lipidology isolation, analysis and identification of lipids, 2nd ed. rev., pp. 106–107, 241–246. Amsterdam: Elsevier. Kim, B.-Y., Weon, H.-Y., Yoo, S.-H., Chen, W.-M., Kwon, S.-W., Go, S.-J. & Stackebrandt, E. (2006). Chitinimonas koreensis sp. nov.,
isolated from greenhouse soil in Korea. Int J Syst Evol Microbiol 56, 1761–1764. Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Jeon, Y. S., Lee, J. H. & other authors (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. Li, Y., Bai, S., Yang, C., Lai, Q., Zhang, H., Chen, Z., Wei, J., Zheng, W., Tian, Y. & Zheng, T. (2013a). Mangrovimonas yunxiaonensis gen. nov.,
sp. nov., isolated from mangrove sediment. Int J Syst Evol Microbiol 63, 2043–2048. Li, Y., Wei, J., Yang, C., Lai, Q., Chen, Z., Li, D., Zhang, H., Tian, Y., Zheng, W. & Zheng, T. (2013b). Tenacibaculum xiamenense sp. nov.,
Acknowledgements This work was supported by the Public Science and Technology Research Funds Projects of Ocean (201305016, 201305022), National Nature Science Foundation of China (40930847, 41376119) and Science and Technology Innovation Funds of Shenzhen (JCYJ20120615161239998). We would like to thank Professor I. J. Hodgkiss from Hong Kong University for help with English.
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