International Journal of Systematic and Evolutionary Microbiology (2015), 65, 418–423
DOI 10.1099/ijs.0.067249-0
Chitinophaga longshanensis sp. nov., a mineral-weathering bacterium isolated from weathered rock Shan Gao, Wen-Bin Zhang, Xia-Fang Sheng, Lin-Yan He and Zhi Huang Correspondence Xia-Fang Sheng [email protected]
Laboratory of Agricultural and Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, PR China A Gram-stain-negative, aerobic, yellow-pigmented, non-motile, non-spore-forming, rod-shaped bacterial strain, Z29T, was isolated from the surface of weathered rock (potassic trachyte) from Nanjing, Jiangsu Province, PR China. Phylogenetic analysis based on 16S rRNA gene sequences suggested that strain Z29T belongs to the genus Chitinophaga in the family Chitinophagaceae. Levels of 16S rRNA gene sequence similarity between strain Z29T and the type strains of recognized species of the genus Chitinophaga ranged from 92.7 to 98.2 %. The main fatty acids of strain Z29T were iso-C15 : 0, C16 : 1v5c and iso-C17 : 0 3-OH. It also contained menaquinone 7 (MK-7) as the respiratory quinone and homospermidine as the main polyamine. The polar lipid profile contained phosphatidylethanolamine, unknown aminolipids, unknown phospholipids and unknown lipids. The total DNA G+C content of strain Z29T was 51.3 mol%. Phenotypic properties and chemotaxonomic data supported the affiliation of strain Z29T with the genus Chitinophaga. The low level of DNA–DNA relatedness (ranging from 14.6 to 29.8 %) to the type strains of other species of the genus Chitinophaga and differential phenotypic properties demonstrated that strain Z29T represents a novel species of the genus Chitinophaga, for which the name Chitinophaga longshanensis sp. nov. is proposed. The type strain is Z29T (5CCTCC AB 2014066T5LMG 28237T).
The genus Chitinophaga, originally described by Sangkhobol & Skerman (1981), is the type genus of the family Chitinophagaceae, which was recently proposed by Ka¨mpfer et al. (2011). Ka¨mpfer et al. (2006) reclassified four species, [Flexibacter] sancti, [Flexibacter] filiformis, [Flexibacter] japonensis and [Cytophaga] arvensicola, to the genus Chitinophaga and described a novel species, Chitinophaga skermanii. Species of the genus Chitinophaga have mostly been isolated from roots of different plants such as Arabidopsis thaliana (Lin et al., 2014) and Cymbidium goeringii (Li et al., 2013), rhizosphere soils and weathered rocks (Wang et al., 2014). Cells of members of the genus are Gram-negative, nonmotile, non-spore-forming and rod-shaped. At the time of writing, there are 19 species with validly published names in the genus Chitinophaga, including the recently described Chitinophaga jiangningensis (Wang et al., 2014), Chitinophaga polysaccharea (Han et al., 2014), Chitinophaga taiwanensis (Lin et al., 2014) and Chitinophaga costaii (Proenc¸a et al., 2014). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Z29T is KJ579707. Five supplementary figures are available with the online Supplementary Material.
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Strain Z29T was isolated while investigating the bacterial diversity on the surfaces of weathered rocks (potassic trachyte) in Nanjing, China. The dilution plating method with a sucrose–mineral salts medium was used to isolate bacterial strains. The medium for isolation contained (per litre): 10.0 g sucrose, 0.5 g yeast extract, 1 g (NH4)2SO4, 2 g K2HPO4, 0.5 g MgSO4, 0.1 g NaCl, 0.5 g CaCO3 and 20 g agar. Weathered rock samples were added to flasks containing physiological salt solution (0.85 %, w/v, NaCl) and shaken at 150 r.p.m. for 30 min to allow bacteria to detach from the rock particles. The suspensions were then allowed to stand for about 10 min. Serial 10-fold dilutions of sample suspensions (1023–1025) were plated onto agar plates to determine total culturable bacteria. The plates were incubated for 3 days at 28 uC. Strain Z29T was able to weather two silicate minerals, biotite and feldspar, and to release Fe, Si and Al from them. Mineral dissolution experiments showed that the Fe, Si and Al released by strain Z29T from the minerals were increased by 3- to 76fold, 1.2- to 5-fold and 7- to 15-fold, respectively, compared with uninoculated controls. The strain was routinely cultured on R2A agar media (0.5 g yeast extract, 0.5 g proteose peptone No. 3, 0.5 g casamino acids, 0.5 g glucose, 0.5 g soluble starch, 0.3 g sodium pyruvate, 0.3 g K2HPO4, 0.05 g 067249 G 2015 IUMS Printed in Great Britain
Chitinophaga longshanensis sp. nov.
MgSO4?7H2O, 15.0 g agar in 1.0 L distilled water. pH7.2) for additional taxonomic experiments. The strain was maintained as a glycerol suspension (20 %, v/v) at 280 uC. Cellular morphology and the presence of flagella were examined by light microscopy (CX21; Olympus) and transmission electron microscopy (H-7650; Hitachi) using exponential-phase cells grown in R2A liquid medium (Huang et al., 2012). Gram staining was determined as described by Murray et al. (1994). Cell growth was tested in R2A liquid medium at different temperatures (4, 15, 20, 25, 28, 30, 37, 42 and 45 uC) and at various pH values (pH 4.0–10.0, at intervals of 0.5 pH units), and was assayed after 5 days of incubation. The buffers that were used to adjust the pH of the R2A medium (each at a final concentration of 100 mM) were acetate (for pH 4.0–5.0), phosphate (for pH 6.0–8.0) and Tris (for pH 9.0–10.0). Salt tolerance was tested in R2A broth supplemented with 0–5 % (w/v) NaCl (at 1.0 % intervals) also after 5 days of incubation at 30 uC. Growth on Luria–Bertani (LB) agar (10.0 g tryptone, 5.0 g yeast extract, 10.0 g NaCl, 15.0 g agar in 1.0 L distilled water. pH7.0), nutrient agar (NA) (10.0 g peptone, 10.0 g beef extract, 5.0 g NaCl, 15.0 g agar in 1.0 L distilled water. pH7.0–7.2), tripticase soy agar (TSA) (15.0 g pancreatic digest of casein, 5.0 g papaic digest of soybean meal, 5.0 g NaCl, 15.0 g agar in 1.0 L distilled water. pH 7.3), R2A agar and MacConkey agar (17.0 g peptone, 3.0 g proteose peptone, 10.0 g lactose, 1.5 g bile salts, 5.0 g NaCl, 0.03 g neutral red, 0.001 g crystal violet, 13.5 g agar in 1.0 L distilled water. pH 7.1) was evaluated at 30 uC after 5 days of incubation. The presence of flexirubin-like pigments was investigated by flooding the plates with 20 % (w/v) potassium hydroxide (Fautz & Reichenbach, 1980). Gliding motility was tested using the hanging drop technique as described by Bernardet et al. (2002). Catalase activity was determined by assessing bubble production by cells in 3 % (v/v) H2O2 and oxidase activity was determined by using 1 % (w/v) N,N-dimethyl-p-phenylenediamine dihydrochloride (bioMe´rieux). Carbon source utilization was determined by using Biolog GN2 MicroPlates as recommended by the manufacturer. Enzyme activities and acid production from different carbohydrates were determined by using API ZYM and API 20NE kits (bioMe´rieux), respectively, according to the manufacturer’s instructions. API 50 CH kits (bioMe´rieux) with minimal medium were also used to determine the strain’s ability to assimilate different carbon sources and to produce acids, again according to the manufacturer’s instructions. Strain Z29T stained Gram-negative and cells were nonmotile, non-spore-forming and short rods with a length of 2.0–3.0 mm and width of 0.8–1.0 mm (Fig. S1, available in the online Supplementary Material). Colonies grown on LB and R2A agar for 48 h were circular, smooth and deep yellow. The strain grew well on LB agar, NA agar, TSA and R2A agar, but did not grow on MacConkey agar. In R2A liquid medium, it was able to grow at 4–40 uC (optimum 30 uC), at pH 5.0–10.0 (optimum pH 7.0) and with 0–2 % http://ijs.sgmjournals.org
(w/v) NaCl (optimum 1 %). More specific physiological characteristics of strain Z29T are given in the species description and a comparison of different characteristics between strain Z29T and other related type strains is given in Table 1. For the comparative analysis of fatty acids, cells of strain Z29T and other reference strains were harvested from R2A agar plates after incubation for 48 h at 30 uC. Cellular fatty acids were extracted and methylated according to the protocol of Miller (1982). The fatty acid methyl ester mixtures were separated using the Sherlock Microbial Identification System (MIS) (MIDI), which consisted of a gas chromatograph (6890N; Agilent) fitted with a 5 % phenylmethylsilicone capillary column (0.2 mm625 m), a flame-ionization detector, an automatic sampler (7683A; Agilent) and a computer (Hewlett Packard). The results were compared with the MIDI database (version 6.1) (Sasser, 1990). Polar lipids of the strain were extracted as described by Minnikin et al. (1984) and separated by two-dimensional TLC on Merck Kieselgel 60-HPTLC plates. Total polar lipids were revealed by spraying with phosphomolybdic acid solution (SigmaAldrich) followed by heating at 150 uC for 10 min, and other plates were sprayed with ninhydrin for aminolipids (Ross et al., 1985) and 1-naphthol spray reagent for glycolipids (Jacin & Mishkin, 1965). Isoprenoid quinones were extracted and purified as described by Collins et al. (1977) and Tamaoka et al. (1983) and then analysed by HPLC (1100; Agilent) with a Zorbax EclipseXBD-C18.5 column (25064.6 mm). Polyamines were extracted as described by Busse & Auling (1988). HPLC was used for analysis (Waters) with a Zorbax Eclipse Plus C18 column (1.8 mm, 10062.1 mm i.d.). The cellular fatty acid profiles of strain Z29T and the type strains of related species of the genus Chitinophaga are shown in Table 2. The major cellular fatty acids of strain Z29T were iso-C15 : 0, C16 : 1v5c and iso-C17 : 0 3-OH. C14 : 0 was not detected in Chitinophaga polysaccharea KACC 17184T, while iso-C16 : 0 was detected only in strain Z29T and Chitinophaga polysaccharea KACC 17184T and Chitinophaga arvensicola JCM 2836T. Furthermore, C18 : 1v9c and cyclo-C19 : 0v8c were only detected in strain Z29T and Chitinophaga polysaccharea KACC 17184T, respectively (Table 2). Strain Z29T exhibited a polar lipid profile consisting of phosphatidylethanolamine as the major component, together with three unknown aminolipids, two unknown phospholipids and four unknown lipids (Fig. S2). Although strain Z29T shared components with Chitinophaga polysaccharea KACC 17184T, unknown aminophospholipids were detected only in Chitinophaga polysaccharea KACC 17184T but not in strain Z29T (Fig. S2). The respiratory quinone detected in strain Z29T was menaquinone 7 (MK-7). The respiratory quinones of Chitinophaga arvensicola JCM 2836T were MK-7 (61.5 %) and MK-7(H4) (38.5 %). Homospermidine was the major polyamine detected in strain Z29T and in Chitinophaga polysaccharea KACC 17184T, while spermidine and putrescine were the minor polyamines (,1 %) detected in strain 419
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Table 1. Differential characteristics of strain Z29T and other related strains belonging to the genus Chitinophaga Strains: 1, Z29T; 2, Chitinophaga polysaccharea KACC 17184T; 3, Chitinophaga arvensicola JCM 2836T; 4, Chitinophaga ginsengisegetis LMG 23601T; 5, Chitinophaga niastensis JCM 15441T; 6, Chitinophaga taiwanensis JCM 18895T. All data were obtained in this study. All strains were able to utilize dextrin, cellobiose, D-galactose, gentiobiose, a-D-glucose, a-lactose, maltose, D-mannose, melibiose, methyl b-D-glucoside, trehalose, turanose, Lasparagine, L-aspartic acid, L-glutamic acid and L-proline, but not a-cyclodextrin, glycogen, Tween 80, D-galactonic acid lactone, D-galacturonic acid, a-hydroxybutyric acid, a-ketobutyric acid, propionic acid, sebacic acid, succinamic acid, glucuronamide, D-alanine, L-leucine, L-pyroglutamic acid, D-serine, L-threonine, inosine, phenyethylamine or 2-aminoethanol as carbon resources. All strains assimilated D-glucose, aesculin ferric citrate, 4-nitophenyl b-D-galactopyranoside, D-mannose, L-arabinose, D-galactose, D-fructose, methyl a-D-mannopyranoside, methyl a-Dglucopyranoside, N-acetylglucosamine, lactose and gentiobiose, but not L-tryptophan, L-arginine, urea, gelatin, capric acid, adipic acid, erythritol, L-xylose, aesculin, glycogen, xylitol, D-arabitol, L-arabitol, potassum 2-ketogluconate or potassum 5-ketogluconate. Based on API ZYM test strips, all strains were positive for alkaline phosphatase, leucine arylamidase, valine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase, but negative for lipase (C14) activity. +, Positive; 2, negative; W, weakly positive. Characteristic Enzyme activities from API ZYM Esterase (C4) a-Fucosidase a-Glucosidase b-Glucosidase Trypsin API 20NE results N-Acetylglucosamine Phenylacetic acid Potassium gluconate Assimilation of (results from API 50 CH): D-Adonitol D-Fucose Inulin Utilization of carbon sources (Biolog) Adonitol D-Gluconic acid Glucose 6-phosphate Itaconic acid a-Ketovaleric acid L-Serine myo-Inositol Putrescine Tween 40 Catalase/oxidase Growth at 37 uC pH range for growth DNA G+C content (mol%)
1
2
3
4
5
6
+ 2 + + +
+ + + + +
2
2
W
W
+ 2
2 2 2
+ + +
+
+ + + +
W
W
2 + +
+ 2 2
+ 2 2
+ 2 2
+ 2 2
+ + +
+ W
2 + W
2 2 +
2 2 2
2
2
2 2 2
+ + 2 + + + + + + +/+ + 5.0–10.0 51.3
+ 2 + 2 2 2 + 2 2 2/2 + 4.0–10.0 47.9
2 2 2 2 2 2 2 2 + +/+ + 4.5–8.0 46.0
2 2 2 2 2 2 2 2 2 2/2 + 5.5–8.5 47.1
2 2 + 2 2 2 2 2 2 +/+ 2 5.0–9.0 43.0
+ + 2 2 + + 2 2 2 +/+ 2 5.0–8.0 53.4
Z29T and Chitinophaga polysaccharea KACC 17184T, respectively (Fig. S3).
W
W
W
The extraction of bacterial genomic DNA was performed using a Qiagen Genomic DNA kit according to the manufacturer’s instructions, which was used as the template for amplifying the 16S rRNA gene. Sequencing of the 16S rRNA gene was performed as described by Timke et al. (2005) using universal primers (27F and 1492R) (Lane, 1991) on an ABI 3730 sequencer (Invitrogen). Phylogenetic analysis was performed using MEGA5 software (Tamura et al., 2011) and three different methods were used to reconstruct phylogenetic trees by bootstrap analyses (Felsenstein, 1985) after 1000 replications: the neighbour-joining (Saitou & Nei,
1987), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Fitch, 1971) algorithms. The algorithm of Kimura’s two-parameter model (Kimura, 1983) was used to calculate evolutionary distance matrices for the neighbour-joining method. The neighbour-joining tree is shown in Fig. 1 and the minimum-likelihood and maximumparsimony trees are available as Figs S4 and S5. The DNA G+C content of strain Z29T was determined by the thermal denaturation method (Marmur & Doty, 1962) using Escherichia coli K-12 as a reference. DNA–DNA hybridizations among strain Z29T, Chitinophaga polysaccharea KACC 17184T, Chitinophaga arvensicola JCM 2836T, Chitinophaga ginsengisegetis LMG 23601T and Chitinophaga niastensis JCM
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Table 2. Cellular fatty acid composition of strain Z29T and the type strains of related species of the genus Chitinophaga Strains: 1, Z29T; 2, Chitinophaga polysaccharea KACC 17184T; 3, Chitinophaga arvensicola JCM 2836T; 4, Chitinophaga ginsengisegetis LMG 23601T; 5, Chitinophaga niastensis JCM 15441T; 6, Chitinophaga taiwanensis CC-ALB-1T. Values represent percentages of total fatty acids. 2, Not detected or lower than 1 %. All data were obtained in this study. Fatty acid C14 : 0 iso-C15 : 0 C16 : 1v5c iso-C16 : 0 C16 : 0 iso-C15 : 0 3-OH C16 : 0 3-OH iso-C17 : 0 3-OH C18 : 1v9c cyclo-C19 : 0v8c C18 : 0 Summed features* 3 8
1
2
3
4
5
6
1.7 29.6 22.9 2.0 8.4 3.4 2.1 10.0 1.9 2 4.0
2 10.7 11.1 1.3 6.9 2.9 2.9 4.5 2 16.9 2.4
1.9 30.4 31.0 1.2 10.2 3.7 2.0 9.5 2 2 2
2.4 31.9 27.5 2 5.2 3.7 2.3 9.9 2 2 1.5
1.9 37.0 32.0 2 4.6 3.8 2.6 8.7 2 2 2
1.7 36.5 25.3 2 7.7 3.7 1.1 12.1 2 2 2
5.9 1.7
2.6 22.0
3.2 2
6.8 1.4
3.9 2
3.6 2
*Summed features represent groups of two or three fatty acids that could not be separated by GLC with the MIDI system. Summed feature 3 comprised C16 : 1v6c and/or C16 : 1v7c; summed feature 8 comprised C18 : 1v6c and/or C16 : 1v7c.
15441T were determined using a UV/VIS spectrophotometer (UV1201; Rayleigh) as described by De Ley et al. (1970). A total of 1449 bp of the 16S rRNA gene of strain Z29T was sequenced. The sequence was subjected to similarity searches using the NCBI BLAST program (http://www.ncbi. nlm.nih.gov), Ribosomal Database Project II (http://rdp. cme.msu.edu/) and the EzBioCloud server (EzTaxon-e database; Kim et al., 2012). The 16S rRNA gene sequence of strain Z29T was aligned with the published sequences of closely related type strains using CLUSTAL W version 2.0 software (Thompson et al., 1997). Comparative 16S rRNA gene sequence analysis showed that strain Z29 T was related most closely to members of the genus Chitinophaga. The 16S rRNA gene sequence of strain Z29T showed similarities of 97.9, 98.2, 98.2, 97.2 and 96.0 % with those of Chitinophaga polysaccharea MRP-15T, Chitinophaga arvensicola M64T, Chitinophaga ginsengisegetis Gsoil 040T, Chitinophaga niastensis JS16-4T and Chitinophaga taiwanensis CCALB-1T, respectively. The genomic DNA G+C content of strain Z29T was 51.3 mol%, which was close to that found for other species of the genus Chitinophaga. DNA–DNA hybridization studies showed relatively low relatedness values between strain Z29T and Chitinophaga polysaccharea KACC 17184T (14.8 %), Chitinophaga arvensicola JCM 2836T (24.4 %), Chitinophaga ginsengisegetis LMG 23601T (29.8 %) and http://ijs.sgmjournals.org
Chitinophaga niastensis JCM 15441T (14.6 %). All of the values were significantly lower than 70 %, the threshold value recommended for the assignment of genomic species (Wayne et al., 1987). These results indicated that strain Z29T represents a novel species of the genus Chitinophaga. Phylogenetic analysis, enzyme activities and differences in other physiological and biochemical characteristics (Table 1) together with the fatty acid profile (Table 2) clearly distinguish strain Z29T from closely related species of the genus Chitinophaga. Thus, on the basis of the data presented, we suggest that strain Z29T represents a novel species of the genus Chitinophaga, for which the name Chitinophaga longshanensis sp. nov. is proposed. Description of Chitinophaga longshanensis sp. nov. Chitinophaga longshanensis (long.shan.en9sis. N.L. fem. adj. longshanensis referring to Xiao Longshan district, Nanjing, Jiangsu Province, PR China, where the type strain was isolated). Cells are Gram-stain-negative, aerobic, non-motile, nonspore-forming rods with a length of 2.0–3.0 mm and width of 0.8–1.0 mm after 20 h of culture on R2A agar. Colonies grown on R2A agar are yellow, circular, slightly convex and sticky. Growth occurs at 4–40 uC and at pH 5.0–10.0, but grows slowly at 4 uC, with optimal growth at 30 uC, at pH 7.0 and with 1 % (w/v) NaCl. Oxidase- and catalasepositive. The major fatty acids (¢10 % of total) are isoC15 : 0, C16 : 1v5c and iso-C17 : 0 3-OH. The polar lipid profile contains phosphatidylethanolamine, unknown aminolipids, unknown phospholipids and unknown lipids. The predominant quinone is MK-7 and the major polyamine is homospermidine. According to the Biolog GN2 system, the following carbon sources are oxidized: dextrin, Tween 40, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, adonitol, L-arabinose, cellobiose, D-fructose, L-fucose, Dgalactose, gentiobiose, a-D-glucose, myo-inositol, a-lactose, lactulose, maltose, D-mannose, melibiose, methyl b-Dglucoside, D-psicose, raffinose, L-rhamnose, sucrose, trehalose, turanose, methyl pyruvate, monomethyl succinate, acetic acid, formic acid, D-gluconic acid, D-glucuronic acid, b-hydroxybutyric acid, c-hydroxybutyric acid, itaconic acid, a-ketovaleric acid, DL-lactic acid, L-asparagine, L-aspartic acid, L-glutamic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-histidine, L-ornithine, L-proline, L-serine, putrescine, 2,3-butanediol, glycerol, DL-a-glycerol phosphate and glucose 1-phosphate. Reduction of nitrates to nitrites and nitrate to nitrogen gas are observed. Assimilates D-glucose, L-arabinose, D-mannose, maltose, potassium gluconate, trisodium citrate, phenylacetic acid, D-adonitol, D-galactose, methyl a-D-glucopyranoside, amygdalin, cellobiose, lactose, melibiose, sucrose, trehalose, raffinose, turanose, D-fructose, L-rhamnose, methyl a-D-mannopyranoside, N-acetylglucosamine, arbutin, salicin, gentiobiose, D-fucose and L-fucose in the API 20NE and API 50 CH systems. In the API ZYM system, positive for alkaline phosphatase, esterase (C4), 421
S. Gao and others
Chitinophaga niastensis JS16-4T (EU714260)
62
0.01
Chitinophaga taiwanensis CC-ALB-1T (KC479802) Chitinophaga ginsegisegetis Gsoil 040T (AB264798) Chitinophaga arvensicola M64T (AM237311)
89
Chitinophaga polysaccharea MRP-15T (KC430923)
100
73
Chitinophaga longshanensis Z29T (KJ579707) Chitinophaga eiseniae YC6729T (FJ750951)
92
84
Chitinophaga terrae KP01T (AB278570) 81
Chitinophaga jiangningensis JN53T (KF150362) Chitinophaga rupis CS5-B1T (FM865977)
99
55
Chitinophaga japonensis 758T (AB078055) Chitinophaga niabensis JS13-10T (EU714259)
97
Chitinophaga cymbidii R156-2T (JN680880) Chitinophaga oryziterrae YC7001T (JF710262)
96
Chitinophaga sancti ATCC 23092T (AB078066) 87
Chitinophaga pinensis ACM 2034T (CP001699) Chitinophaga filiformis Fx el ReichenbachT (AB078049)
100 100
Chitinophaga ginsengisoli Gsoil 052T (AB245374)
Chitinophaga costaii A37T2T (KC922450) Ferruginibacter yonginensis HME8442T (KC690144)
Fig. 1. Rooted neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the position of strain Z29T and species of the genus Chitinophaga. Bootstrap values (expressed as percentages of 1000 replications) only over 50 % are given at branch points. Filled circles at nodes show generic branches that were also recovered by using the maximum-likelihood and maximum-parsimony algorithms. Bar, 0.01 substitutions per nucleotide position.
leucine arylamidase, valine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, b-galactosidase, a-glucosidase, b-glucosidase and N-acetyl-b-glucosaminidase. With minimal medium (50 CHB/E medium) in the API 50 CH system, produces acids from L-arabinose, Dadonitol, D-galactose, D-glucose, D-mannose, methyl a-Dglucopyranoside, amygdalin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, raffinose, turanose, D-fructose, L-rhamnose, methyl a-D-mannopyranoside, N-acetylglucosamine, arbutin, salicin, gentiobiose, D-fucose and L-fucose. The type strain, Z29T (5CCTCC AB 2014066T5LMG 28237T), was isolated from the surfaces of weathered rock in Longshan (Nanjing, China). The DNA G+C content of the type strain is 51.3 mol%.
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DOI 10.1099/ijs.0.067249-0
Chitinophaga longshanensis sp. nov., a mineral-weathering bacterium isolated from weathered rock Shan Gao, Wen-Bin Zhang, Xia-Fang Sheng, Lin-Yan He and Zhi Huang Correspondence Xia-Fang Sheng [email protected]
Laboratory of Agricultural and Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, PR China A Gram-stain-negative, aerobic, yellow-pigmented, non-motile, non-spore-forming, rod-shaped bacterial strain, Z29T, was isolated from the surface of weathered rock (potassic trachyte) from Nanjing, Jiangsu Province, PR China. Phylogenetic analysis based on 16S rRNA gene sequences suggested that strain Z29T belongs to the genus Chitinophaga in the family Chitinophagaceae. Levels of 16S rRNA gene sequence similarity between strain Z29T and the type strains of recognized species of the genus Chitinophaga ranged from 92.7 to 98.2 %. The main fatty acids of strain Z29T were iso-C15 : 0, C16 : 1v5c and iso-C17 : 0 3-OH. It also contained menaquinone 7 (MK-7) as the respiratory quinone and homospermidine as the main polyamine. The polar lipid profile contained phosphatidylethanolamine, unknown aminolipids, unknown phospholipids and unknown lipids. The total DNA G+C content of strain Z29T was 51.3 mol%. Phenotypic properties and chemotaxonomic data supported the affiliation of strain Z29T with the genus Chitinophaga. The low level of DNA–DNA relatedness (ranging from 14.6 to 29.8 %) to the type strains of other species of the genus Chitinophaga and differential phenotypic properties demonstrated that strain Z29T represents a novel species of the genus Chitinophaga, for which the name Chitinophaga longshanensis sp. nov. is proposed. The type strain is Z29T (5CCTCC AB 2014066T5LMG 28237T).
The genus Chitinophaga, originally described by Sangkhobol & Skerman (1981), is the type genus of the family Chitinophagaceae, which was recently proposed by Ka¨mpfer et al. (2011). Ka¨mpfer et al. (2006) reclassified four species, [Flexibacter] sancti, [Flexibacter] filiformis, [Flexibacter] japonensis and [Cytophaga] arvensicola, to the genus Chitinophaga and described a novel species, Chitinophaga skermanii. Species of the genus Chitinophaga have mostly been isolated from roots of different plants such as Arabidopsis thaliana (Lin et al., 2014) and Cymbidium goeringii (Li et al., 2013), rhizosphere soils and weathered rocks (Wang et al., 2014). Cells of members of the genus are Gram-negative, nonmotile, non-spore-forming and rod-shaped. At the time of writing, there are 19 species with validly published names in the genus Chitinophaga, including the recently described Chitinophaga jiangningensis (Wang et al., 2014), Chitinophaga polysaccharea (Han et al., 2014), Chitinophaga taiwanensis (Lin et al., 2014) and Chitinophaga costaii (Proenc¸a et al., 2014). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Z29T is KJ579707. Five supplementary figures are available with the online Supplementary Material.
418
Strain Z29T was isolated while investigating the bacterial diversity on the surfaces of weathered rocks (potassic trachyte) in Nanjing, China. The dilution plating method with a sucrose–mineral salts medium was used to isolate bacterial strains. The medium for isolation contained (per litre): 10.0 g sucrose, 0.5 g yeast extract, 1 g (NH4)2SO4, 2 g K2HPO4, 0.5 g MgSO4, 0.1 g NaCl, 0.5 g CaCO3 and 20 g agar. Weathered rock samples were added to flasks containing physiological salt solution (0.85 %, w/v, NaCl) and shaken at 150 r.p.m. for 30 min to allow bacteria to detach from the rock particles. The suspensions were then allowed to stand for about 10 min. Serial 10-fold dilutions of sample suspensions (1023–1025) were plated onto agar plates to determine total culturable bacteria. The plates were incubated for 3 days at 28 uC. Strain Z29T was able to weather two silicate minerals, biotite and feldspar, and to release Fe, Si and Al from them. Mineral dissolution experiments showed that the Fe, Si and Al released by strain Z29T from the minerals were increased by 3- to 76fold, 1.2- to 5-fold and 7- to 15-fold, respectively, compared with uninoculated controls. The strain was routinely cultured on R2A agar media (0.5 g yeast extract, 0.5 g proteose peptone No. 3, 0.5 g casamino acids, 0.5 g glucose, 0.5 g soluble starch, 0.3 g sodium pyruvate, 0.3 g K2HPO4, 0.05 g 067249 G 2015 IUMS Printed in Great Britain
Chitinophaga longshanensis sp. nov.
MgSO4?7H2O, 15.0 g agar in 1.0 L distilled water. pH7.2) for additional taxonomic experiments. The strain was maintained as a glycerol suspension (20 %, v/v) at 280 uC. Cellular morphology and the presence of flagella were examined by light microscopy (CX21; Olympus) and transmission electron microscopy (H-7650; Hitachi) using exponential-phase cells grown in R2A liquid medium (Huang et al., 2012). Gram staining was determined as described by Murray et al. (1994). Cell growth was tested in R2A liquid medium at different temperatures (4, 15, 20, 25, 28, 30, 37, 42 and 45 uC) and at various pH values (pH 4.0–10.0, at intervals of 0.5 pH units), and was assayed after 5 days of incubation. The buffers that were used to adjust the pH of the R2A medium (each at a final concentration of 100 mM) were acetate (for pH 4.0–5.0), phosphate (for pH 6.0–8.0) and Tris (for pH 9.0–10.0). Salt tolerance was tested in R2A broth supplemented with 0–5 % (w/v) NaCl (at 1.0 % intervals) also after 5 days of incubation at 30 uC. Growth on Luria–Bertani (LB) agar (10.0 g tryptone, 5.0 g yeast extract, 10.0 g NaCl, 15.0 g agar in 1.0 L distilled water. pH7.0), nutrient agar (NA) (10.0 g peptone, 10.0 g beef extract, 5.0 g NaCl, 15.0 g agar in 1.0 L distilled water. pH7.0–7.2), tripticase soy agar (TSA) (15.0 g pancreatic digest of casein, 5.0 g papaic digest of soybean meal, 5.0 g NaCl, 15.0 g agar in 1.0 L distilled water. pH 7.3), R2A agar and MacConkey agar (17.0 g peptone, 3.0 g proteose peptone, 10.0 g lactose, 1.5 g bile salts, 5.0 g NaCl, 0.03 g neutral red, 0.001 g crystal violet, 13.5 g agar in 1.0 L distilled water. pH 7.1) was evaluated at 30 uC after 5 days of incubation. The presence of flexirubin-like pigments was investigated by flooding the plates with 20 % (w/v) potassium hydroxide (Fautz & Reichenbach, 1980). Gliding motility was tested using the hanging drop technique as described by Bernardet et al. (2002). Catalase activity was determined by assessing bubble production by cells in 3 % (v/v) H2O2 and oxidase activity was determined by using 1 % (w/v) N,N-dimethyl-p-phenylenediamine dihydrochloride (bioMe´rieux). Carbon source utilization was determined by using Biolog GN2 MicroPlates as recommended by the manufacturer. Enzyme activities and acid production from different carbohydrates were determined by using API ZYM and API 20NE kits (bioMe´rieux), respectively, according to the manufacturer’s instructions. API 50 CH kits (bioMe´rieux) with minimal medium were also used to determine the strain’s ability to assimilate different carbon sources and to produce acids, again according to the manufacturer’s instructions. Strain Z29T stained Gram-negative and cells were nonmotile, non-spore-forming and short rods with a length of 2.0–3.0 mm and width of 0.8–1.0 mm (Fig. S1, available in the online Supplementary Material). Colonies grown on LB and R2A agar for 48 h were circular, smooth and deep yellow. The strain grew well on LB agar, NA agar, TSA and R2A agar, but did not grow on MacConkey agar. In R2A liquid medium, it was able to grow at 4–40 uC (optimum 30 uC), at pH 5.0–10.0 (optimum pH 7.0) and with 0–2 % http://ijs.sgmjournals.org
(w/v) NaCl (optimum 1 %). More specific physiological characteristics of strain Z29T are given in the species description and a comparison of different characteristics between strain Z29T and other related type strains is given in Table 1. For the comparative analysis of fatty acids, cells of strain Z29T and other reference strains were harvested from R2A agar plates after incubation for 48 h at 30 uC. Cellular fatty acids were extracted and methylated according to the protocol of Miller (1982). The fatty acid methyl ester mixtures were separated using the Sherlock Microbial Identification System (MIS) (MIDI), which consisted of a gas chromatograph (6890N; Agilent) fitted with a 5 % phenylmethylsilicone capillary column (0.2 mm625 m), a flame-ionization detector, an automatic sampler (7683A; Agilent) and a computer (Hewlett Packard). The results were compared with the MIDI database (version 6.1) (Sasser, 1990). Polar lipids of the strain were extracted as described by Minnikin et al. (1984) and separated by two-dimensional TLC on Merck Kieselgel 60-HPTLC plates. Total polar lipids were revealed by spraying with phosphomolybdic acid solution (SigmaAldrich) followed by heating at 150 uC for 10 min, and other plates were sprayed with ninhydrin for aminolipids (Ross et al., 1985) and 1-naphthol spray reagent for glycolipids (Jacin & Mishkin, 1965). Isoprenoid quinones were extracted and purified as described by Collins et al. (1977) and Tamaoka et al. (1983) and then analysed by HPLC (1100; Agilent) with a Zorbax EclipseXBD-C18.5 column (25064.6 mm). Polyamines were extracted as described by Busse & Auling (1988). HPLC was used for analysis (Waters) with a Zorbax Eclipse Plus C18 column (1.8 mm, 10062.1 mm i.d.). The cellular fatty acid profiles of strain Z29T and the type strains of related species of the genus Chitinophaga are shown in Table 2. The major cellular fatty acids of strain Z29T were iso-C15 : 0, C16 : 1v5c and iso-C17 : 0 3-OH. C14 : 0 was not detected in Chitinophaga polysaccharea KACC 17184T, while iso-C16 : 0 was detected only in strain Z29T and Chitinophaga polysaccharea KACC 17184T and Chitinophaga arvensicola JCM 2836T. Furthermore, C18 : 1v9c and cyclo-C19 : 0v8c were only detected in strain Z29T and Chitinophaga polysaccharea KACC 17184T, respectively (Table 2). Strain Z29T exhibited a polar lipid profile consisting of phosphatidylethanolamine as the major component, together with three unknown aminolipids, two unknown phospholipids and four unknown lipids (Fig. S2). Although strain Z29T shared components with Chitinophaga polysaccharea KACC 17184T, unknown aminophospholipids were detected only in Chitinophaga polysaccharea KACC 17184T but not in strain Z29T (Fig. S2). The respiratory quinone detected in strain Z29T was menaquinone 7 (MK-7). The respiratory quinones of Chitinophaga arvensicola JCM 2836T were MK-7 (61.5 %) and MK-7(H4) (38.5 %). Homospermidine was the major polyamine detected in strain Z29T and in Chitinophaga polysaccharea KACC 17184T, while spermidine and putrescine were the minor polyamines (,1 %) detected in strain 419
S. Gao and others
Table 1. Differential characteristics of strain Z29T and other related strains belonging to the genus Chitinophaga Strains: 1, Z29T; 2, Chitinophaga polysaccharea KACC 17184T; 3, Chitinophaga arvensicola JCM 2836T; 4, Chitinophaga ginsengisegetis LMG 23601T; 5, Chitinophaga niastensis JCM 15441T; 6, Chitinophaga taiwanensis JCM 18895T. All data were obtained in this study. All strains were able to utilize dextrin, cellobiose, D-galactose, gentiobiose, a-D-glucose, a-lactose, maltose, D-mannose, melibiose, methyl b-D-glucoside, trehalose, turanose, Lasparagine, L-aspartic acid, L-glutamic acid and L-proline, but not a-cyclodextrin, glycogen, Tween 80, D-galactonic acid lactone, D-galacturonic acid, a-hydroxybutyric acid, a-ketobutyric acid, propionic acid, sebacic acid, succinamic acid, glucuronamide, D-alanine, L-leucine, L-pyroglutamic acid, D-serine, L-threonine, inosine, phenyethylamine or 2-aminoethanol as carbon resources. All strains assimilated D-glucose, aesculin ferric citrate, 4-nitophenyl b-D-galactopyranoside, D-mannose, L-arabinose, D-galactose, D-fructose, methyl a-D-mannopyranoside, methyl a-Dglucopyranoside, N-acetylglucosamine, lactose and gentiobiose, but not L-tryptophan, L-arginine, urea, gelatin, capric acid, adipic acid, erythritol, L-xylose, aesculin, glycogen, xylitol, D-arabitol, L-arabitol, potassum 2-ketogluconate or potassum 5-ketogluconate. Based on API ZYM test strips, all strains were positive for alkaline phosphatase, leucine arylamidase, valine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase, but negative for lipase (C14) activity. +, Positive; 2, negative; W, weakly positive. Characteristic Enzyme activities from API ZYM Esterase (C4) a-Fucosidase a-Glucosidase b-Glucosidase Trypsin API 20NE results N-Acetylglucosamine Phenylacetic acid Potassium gluconate Assimilation of (results from API 50 CH): D-Adonitol D-Fucose Inulin Utilization of carbon sources (Biolog) Adonitol D-Gluconic acid Glucose 6-phosphate Itaconic acid a-Ketovaleric acid L-Serine myo-Inositol Putrescine Tween 40 Catalase/oxidase Growth at 37 uC pH range for growth DNA G+C content (mol%)
1
2
3
4
5
6
+ 2 + + +
+ + + + +
2
2
W
W
+ 2
2 2 2
+ + +
+
+ + + +
W
W
2 + +
+ 2 2
+ 2 2
+ 2 2
+ 2 2
+ + +
+ W
2 + W
2 2 +
2 2 2
2
2
2 2 2
+ + 2 + + + + + + +/+ + 5.0–10.0 51.3
+ 2 + 2 2 2 + 2 2 2/2 + 4.0–10.0 47.9
2 2 2 2 2 2 2 2 + +/+ + 4.5–8.0 46.0
2 2 2 2 2 2 2 2 2 2/2 + 5.5–8.5 47.1
2 2 + 2 2 2 2 2 2 +/+ 2 5.0–9.0 43.0
+ + 2 2 + + 2 2 2 +/+ 2 5.0–8.0 53.4
Z29T and Chitinophaga polysaccharea KACC 17184T, respectively (Fig. S3).
W
W
W
The extraction of bacterial genomic DNA was performed using a Qiagen Genomic DNA kit according to the manufacturer’s instructions, which was used as the template for amplifying the 16S rRNA gene. Sequencing of the 16S rRNA gene was performed as described by Timke et al. (2005) using universal primers (27F and 1492R) (Lane, 1991) on an ABI 3730 sequencer (Invitrogen). Phylogenetic analysis was performed using MEGA5 software (Tamura et al., 2011) and three different methods were used to reconstruct phylogenetic trees by bootstrap analyses (Felsenstein, 1985) after 1000 replications: the neighbour-joining (Saitou & Nei,
1987), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Fitch, 1971) algorithms. The algorithm of Kimura’s two-parameter model (Kimura, 1983) was used to calculate evolutionary distance matrices for the neighbour-joining method. The neighbour-joining tree is shown in Fig. 1 and the minimum-likelihood and maximumparsimony trees are available as Figs S4 and S5. The DNA G+C content of strain Z29T was determined by the thermal denaturation method (Marmur & Doty, 1962) using Escherichia coli K-12 as a reference. DNA–DNA hybridizations among strain Z29T, Chitinophaga polysaccharea KACC 17184T, Chitinophaga arvensicola JCM 2836T, Chitinophaga ginsengisegetis LMG 23601T and Chitinophaga niastensis JCM
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International Journal of Systematic and Evolutionary Microbiology 65
Chitinophaga longshanensis sp. nov.
Table 2. Cellular fatty acid composition of strain Z29T and the type strains of related species of the genus Chitinophaga Strains: 1, Z29T; 2, Chitinophaga polysaccharea KACC 17184T; 3, Chitinophaga arvensicola JCM 2836T; 4, Chitinophaga ginsengisegetis LMG 23601T; 5, Chitinophaga niastensis JCM 15441T; 6, Chitinophaga taiwanensis CC-ALB-1T. Values represent percentages of total fatty acids. 2, Not detected or lower than 1 %. All data were obtained in this study. Fatty acid C14 : 0 iso-C15 : 0 C16 : 1v5c iso-C16 : 0 C16 : 0 iso-C15 : 0 3-OH C16 : 0 3-OH iso-C17 : 0 3-OH C18 : 1v9c cyclo-C19 : 0v8c C18 : 0 Summed features* 3 8
1
2
3
4
5
6
1.7 29.6 22.9 2.0 8.4 3.4 2.1 10.0 1.9 2 4.0
2 10.7 11.1 1.3 6.9 2.9 2.9 4.5 2 16.9 2.4
1.9 30.4 31.0 1.2 10.2 3.7 2.0 9.5 2 2 2
2.4 31.9 27.5 2 5.2 3.7 2.3 9.9 2 2 1.5
1.9 37.0 32.0 2 4.6 3.8 2.6 8.7 2 2 2
1.7 36.5 25.3 2 7.7 3.7 1.1 12.1 2 2 2
5.9 1.7
2.6 22.0
3.2 2
6.8 1.4
3.9 2
3.6 2
*Summed features represent groups of two or three fatty acids that could not be separated by GLC with the MIDI system. Summed feature 3 comprised C16 : 1v6c and/or C16 : 1v7c; summed feature 8 comprised C18 : 1v6c and/or C16 : 1v7c.
15441T were determined using a UV/VIS spectrophotometer (UV1201; Rayleigh) as described by De Ley et al. (1970). A total of 1449 bp of the 16S rRNA gene of strain Z29T was sequenced. The sequence was subjected to similarity searches using the NCBI BLAST program (http://www.ncbi. nlm.nih.gov), Ribosomal Database Project II (http://rdp. cme.msu.edu/) and the EzBioCloud server (EzTaxon-e database; Kim et al., 2012). The 16S rRNA gene sequence of strain Z29T was aligned with the published sequences of closely related type strains using CLUSTAL W version 2.0 software (Thompson et al., 1997). Comparative 16S rRNA gene sequence analysis showed that strain Z29 T was related most closely to members of the genus Chitinophaga. The 16S rRNA gene sequence of strain Z29T showed similarities of 97.9, 98.2, 98.2, 97.2 and 96.0 % with those of Chitinophaga polysaccharea MRP-15T, Chitinophaga arvensicola M64T, Chitinophaga ginsengisegetis Gsoil 040T, Chitinophaga niastensis JS16-4T and Chitinophaga taiwanensis CCALB-1T, respectively. The genomic DNA G+C content of strain Z29T was 51.3 mol%, which was close to that found for other species of the genus Chitinophaga. DNA–DNA hybridization studies showed relatively low relatedness values between strain Z29T and Chitinophaga polysaccharea KACC 17184T (14.8 %), Chitinophaga arvensicola JCM 2836T (24.4 %), Chitinophaga ginsengisegetis LMG 23601T (29.8 %) and http://ijs.sgmjournals.org
Chitinophaga niastensis JCM 15441T (14.6 %). All of the values were significantly lower than 70 %, the threshold value recommended for the assignment of genomic species (Wayne et al., 1987). These results indicated that strain Z29T represents a novel species of the genus Chitinophaga. Phylogenetic analysis, enzyme activities and differences in other physiological and biochemical characteristics (Table 1) together with the fatty acid profile (Table 2) clearly distinguish strain Z29T from closely related species of the genus Chitinophaga. Thus, on the basis of the data presented, we suggest that strain Z29T represents a novel species of the genus Chitinophaga, for which the name Chitinophaga longshanensis sp. nov. is proposed. Description of Chitinophaga longshanensis sp. nov. Chitinophaga longshanensis (long.shan.en9sis. N.L. fem. adj. longshanensis referring to Xiao Longshan district, Nanjing, Jiangsu Province, PR China, where the type strain was isolated). Cells are Gram-stain-negative, aerobic, non-motile, nonspore-forming rods with a length of 2.0–3.0 mm and width of 0.8–1.0 mm after 20 h of culture on R2A agar. Colonies grown on R2A agar are yellow, circular, slightly convex and sticky. Growth occurs at 4–40 uC and at pH 5.0–10.0, but grows slowly at 4 uC, with optimal growth at 30 uC, at pH 7.0 and with 1 % (w/v) NaCl. Oxidase- and catalasepositive. The major fatty acids (¢10 % of total) are isoC15 : 0, C16 : 1v5c and iso-C17 : 0 3-OH. The polar lipid profile contains phosphatidylethanolamine, unknown aminolipids, unknown phospholipids and unknown lipids. The predominant quinone is MK-7 and the major polyamine is homospermidine. According to the Biolog GN2 system, the following carbon sources are oxidized: dextrin, Tween 40, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, adonitol, L-arabinose, cellobiose, D-fructose, L-fucose, Dgalactose, gentiobiose, a-D-glucose, myo-inositol, a-lactose, lactulose, maltose, D-mannose, melibiose, methyl b-Dglucoside, D-psicose, raffinose, L-rhamnose, sucrose, trehalose, turanose, methyl pyruvate, monomethyl succinate, acetic acid, formic acid, D-gluconic acid, D-glucuronic acid, b-hydroxybutyric acid, c-hydroxybutyric acid, itaconic acid, a-ketovaleric acid, DL-lactic acid, L-asparagine, L-aspartic acid, L-glutamic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-histidine, L-ornithine, L-proline, L-serine, putrescine, 2,3-butanediol, glycerol, DL-a-glycerol phosphate and glucose 1-phosphate. Reduction of nitrates to nitrites and nitrate to nitrogen gas are observed. Assimilates D-glucose, L-arabinose, D-mannose, maltose, potassium gluconate, trisodium citrate, phenylacetic acid, D-adonitol, D-galactose, methyl a-D-glucopyranoside, amygdalin, cellobiose, lactose, melibiose, sucrose, trehalose, raffinose, turanose, D-fructose, L-rhamnose, methyl a-D-mannopyranoside, N-acetylglucosamine, arbutin, salicin, gentiobiose, D-fucose and L-fucose in the API 20NE and API 50 CH systems. In the API ZYM system, positive for alkaline phosphatase, esterase (C4), 421
S. Gao and others
Chitinophaga niastensis JS16-4T (EU714260)
62
0.01
Chitinophaga taiwanensis CC-ALB-1T (KC479802) Chitinophaga ginsegisegetis Gsoil 040T (AB264798) Chitinophaga arvensicola M64T (AM237311)
89
Chitinophaga polysaccharea MRP-15T (KC430923)
100
73
Chitinophaga longshanensis Z29T (KJ579707) Chitinophaga eiseniae YC6729T (FJ750951)
92
84
Chitinophaga terrae KP01T (AB278570) 81
Chitinophaga jiangningensis JN53T (KF150362) Chitinophaga rupis CS5-B1T (FM865977)
99
55
Chitinophaga japonensis 758T (AB078055) Chitinophaga niabensis JS13-10T (EU714259)
97
Chitinophaga cymbidii R156-2T (JN680880) Chitinophaga oryziterrae YC7001T (JF710262)
96
Chitinophaga sancti ATCC 23092T (AB078066) 87
Chitinophaga pinensis ACM 2034T (CP001699) Chitinophaga filiformis Fx el ReichenbachT (AB078049)
100 100
Chitinophaga ginsengisoli Gsoil 052T (AB245374)
Chitinophaga costaii A37T2T (KC922450) Ferruginibacter yonginensis HME8442T (KC690144)
Fig. 1. Rooted neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the position of strain Z29T and species of the genus Chitinophaga. Bootstrap values (expressed as percentages of 1000 replications) only over 50 % are given at branch points. Filled circles at nodes show generic branches that were also recovered by using the maximum-likelihood and maximum-parsimony algorithms. Bar, 0.01 substitutions per nucleotide position.
leucine arylamidase, valine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, b-galactosidase, a-glucosidase, b-glucosidase and N-acetyl-b-glucosaminidase. With minimal medium (50 CHB/E medium) in the API 50 CH system, produces acids from L-arabinose, Dadonitol, D-galactose, D-glucose, D-mannose, methyl a-Dglucopyranoside, amygdalin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, raffinose, turanose, D-fructose, L-rhamnose, methyl a-D-mannopyranoside, N-acetylglucosamine, arbutin, salicin, gentiobiose, D-fucose and L-fucose. The type strain, Z29T (5CCTCC AB 2014066T5LMG 28237T), was isolated from the surfaces of weathered rock in Longshan (Nanjing, China). The DNA G+C content of the type strain is 51.3 mol%.
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