Pullulanibacillus pueri sp. nov., isolated from Pu er tea

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1 International Journal of Systematic and Evolutionary Microbiology (2015), 65, DOI /ijs Pullulanibacillus pueri sp. nov., isolated from Pu er tea Lili Niu, 1 Tianyi Tang, 1 Lei Song, 2 Mengjie Xiong, 1 Jianqing Tian, 3 Kegui Zhang, 4 Xing Hu 5 and Daochen Zhu 6 Correspondence Lili Niu lilyniu@126.com Daochen Zhu dczhucn@hotmail.com 1 Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, Shanghai , PR China 2 China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing 101, PR China 3 State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 101, PR China 4 School of Life Sciences, Huainan Normal University, Huainan , PR China 5 School of Environmental and Chemical Engineering, Shanghai University, Shanghai , PR China 6 School of Environmental Engineering, Jiangsu University, Zhenjiang , PR China A novel Gram-stain-positive, aerobic, endospore-forming, rod-shaped bacterial strain YN3 T was isolated from ripened Pu er tea. Phylogenetic analysis of 16S rrna gene sequences showed that the strain belonged to the family Sporolactobacillaceae and was closely related to Pullulanibacillus naganoensis DSM T (95.8 % 16S rrna gene sequence similarity) and Pullulanibacillus uraniitolerans DSM T (95.4 %). Growth of the strain was observed at C (optimum C), at ph (optimum ph ). The strain had a cell-wall type A1c peptidoglycan with meso-diaminopimelic acid as the diagnostic diamino acid. The predominant menaquinone was menaquinone-7 (MK-7). The major fatty acids were anteiso-c 15:0, anteiso-c 17:0 and C 18:1 v7c. The DNA G+C content of strain YN3 T was 38.7 mol%. Strain YN3 T could be differentiated from recognized species of the genus Pullulanibacillus based on phenotypic characteristics, chemotaxonomic differences, phylogenetic analysis and DNA DNA hybridization data. On the basis of polyphasic evidence from this study, Pullulanibacillus pueri sp. nov., is proposed, with strain YN3 T (5CGMCC T 5JCM T ) as the type strain. The genus Pullulanibacillus was erected by Hatayama et al. (2006) with Pullulanibacillus naganoensis as the type species on the basis of reclassification of Bacillus naganoensis, and the description was later emended by Pereira et al. (2013). At the time of writing, the genus Pullulanibacillus comprised only two species, P. naganoensis and Pullulanibacillus uraniitolerans ( Members of the genus are Gram-positive, endospore-forming rods. Chemotaxonomic characteristics of the genus include the major menaquinone-7 (MK-7), the major cellar fatty acids iso- and anteiso-branched and a DNA G+C content Abbreviations: ML, maximum-likelihood; MP, maximum-parsimony; NJ, neighbour-joining. The GenBank/EMBL/DDBJ accession number for the 16S rrna gene sequence of strain YN3 T is KF Three supplementary figures and two supplementary tables are available with the online Supplementary Material. of 39 mol%. Strains belonging to the genus Pullulanibacillus have been isolated from soil and uranium mine tailing effluent (Tomimura et al., 1990; Pereira et al., 2013; Hatayama et al., 2006). During the course of a study of bacteria related to the ageing of Pu er tea, a novel strain, designated YN3 T, was isolated from a ripened Pu er tea sample obtained from a manufacturer in Puer City, Yunnan province, China, which had been stored (post-fermented) for six years. Strain YN3 T was characterized using a polyphasic approach, including phylogenetic analyses based on 16S rrna gene sequences, DNA DNA hybridization, chemotaxonomic and phenotypic properties in order to determine its precise taxonomic position. The results indicate that YN3 T is a member of the genus Pullulanibacillus, but it is clearly distinguished from the current species of the genus Pullulanibacillus with validly published names. P. naganoensis DSM T and P. uraniitolerans DSM T were purchased from the Leibniz-Institut Deutsche G 2015 IUMS Printed in Great Britain 2167

2 L. Niu and others Sammlung von Mikroorganismen und Zellkulturen (DSMZ). Strain YN3 T was isolated by plating serial dilutions onto Luria Bertani (LB, laboratory-prepared) agar medium. Single colonies were purified by transferring them onto new plates four times and subjecting them to additional incubation in Alicyclobacillus medium [per litre distilled water: 0.5 g glucose, 0.25 g CaCl 2.2H 2 O, 0.5 g MgSO 4.7H 2 O, 0.2 g (NH 4 ) 2 SO 4, 3.0 g KH 2 PO 4 and 1 ml trace salt solution (per litre: 0.1 g ZnSO 4.7H 2 O, 0.03 g MnCl 2.4H 2 O, 0.3 g H 3 BO 3, 0.2 g CoCl 2.6H 2 O, 0.01 g CuCl 2.2H 2 O, 0.02 g NiCl 6.6H 2 O, 0.03 g NaMoO 4. 2H 2 O); ph ] for 2daysat308C. The purity of strain YN3 T was confirmed by the homogeneous morphology of colonies on agar surface as well as cell type observed under microscope. A pure culture was collected and then preserved at 280 8C as a suspension in Alicyclobacillus medium broth supplemented with 17 % (w/v) glycerol. Light and electron microscopy and analytical techniques were performed as described previously (Niu et al., 2008). The Gram reaction was carried out according to the classical Gram-staining procedure described by Doetsch (1981) and the non-staining KOH method as described by Gregersen (1978). The structure of the cell wall was confirmed by electron microscopy of ultrathin sections. Hydrolysis of casein, starch, gelatin, pectin, xylan, CM-cellulose and Tweens 20, 60 and 80 were assessed according to the protocol of Dong & Cai (2001). The ability to hydrolyse pullulan was examined as described by Pereira et al. (2013). Substrate utilization studies were performed in Alicyclobacillus basal medium containing different substrates as follows: sugars (0.5 %, final concentration), fatty acids (20 mm, final concentration) and amino acids (0.2 %, final concentration). The basal medium contained (l 21 ): 0.25 g CaCl 2.2H 2 O, 0.5 g MgSO 4.7H 2 O, 0.2 g (NH 4 ) 2 SO 4, 3.0 g KH 2 PO 4 and 1 ml trace salt solution (described above). Physiological studies on temperature, ph and salinity ranges for growth were carried out in CYC medium broth [33.4 g Czapek Dox liquid medium, modified (Oxoid), 2.0 g Bacto yeast extract (Difco), 6.0 g Bacto vitamin assay Casamino acids (Difco), 0 ml distilled water, ph 7.0] (Hatayama et al., 2006). Additional enzymic activities were obtained using the API ZYM system (biomérieux) at 30 8C according to the manufacturer s instructions. All tests were performed in duplicate. Cells of strain YN3 T were Gram-stain-positive, straight or slightly curved rods, mm mm in size. Cells occurred singly or in clusters, were peritrichously flagellated and exhibited slight twitching motility. Subterminal, elliptical spores were occasionally formed, which swelled the cells at one terminal. (Figs S1 and S2, available in the online Supplementary Material). Colonies on CYC agar were yellow, circular and slightly convex, with a diameter of 3.0 mm after cultivation at 30 8C for48h. Strain YN3 T grew strictly aerobically and growth occurred from C and at ph , with optimum growth at C and ph , respectively. The strain could grow in the presence of % (w/v) NaCl. Additional physiological and biochemical characteristics of the strain are summarized in the species description, and a comparison of selected characteristics with related type strains is shown in Table 1 and Table S1. Genomic DNA was extracted and purified using the method of Marmur (1961). The 16S rrna gene amplification, sequencing and sequence analysis were done as described previously (Niu et al., 2008). To ascertain the phylogenetic position of strain YN3 T, the complete 16S rrna gene sequence (1553 bp) was compared with the most similar sequences from the GenBank database. All sequence alignments were analysed with the MEGA6 software package (Tamura et al., 2013). For maximum-likelihood (ML) analysis, the best-fit model for nucleotide substitution was selected from 24 models using MEGA6 based on the minimum Bayesian information criterion value. The best model in this study was the Tamura Nei model with gamma-distributed rates plus invariant sites. The ML tree was built using the best-fit model and nearest-neighbour interchange for the ML heuristic method. For neighbour-joining (NJ) trees, the mean pairwise Jukes Cantor distance was chosen to determine whether the sequence data were suitable for estimating NJ trees. The mean distance in this study was 0.112, a value suitable for making NJ trees (Nei & Kumar, 2008). The NJ tree was built using the p-distance method. For maximum-parsimony (MP) analysis, the subtree pruning-regrafting method was used with the default setting of 10 trees. All ML, NJ and MP trees were built with partial deletion of gaps, and reliability of the phylogenetic trees was estimated using bootstrap values based on 0 iterations. The three trees showed generally the same topology, and therefore only the ML tree is shown (Fig. 1). The phylogenetic analyses of strain YN3 T revealed that it was associated with the family Sporolactobacillaceae and was most closely related to members of the genus Pullulanibacillus. The 16S rrna gene sequence similarity values between strain YN3 T and the type strains of related species of the genus Pullulanibacillus were 95.8 % and 95.4 % for P. naganoensis and P. uraniitolerans, respectively. DNA DNA hybridization was carried out as described by De Ley et al. (1970) incorporating the modifications described by Huss et al. (1983), using a Cary Bio UV/VIS-spectrophotometer equipped with a Peltier temperature-controlling programmer. The DNA DNA relatedness values between strain YN3 T and P. naganoensis DSM T and P. uraniitolerans DSM T were 17.3 % and 15.8 %, respectively. These values were wellbelow the threshold value (70 %) recommended by Wayne et al. (1987) for the definition of members of a species. The phylogenetic analysis results and DNA DNA relatedness values described above indicated that the new isolate YN3 T might represent a novel species of the genus Pullulanibacillus. The G+C content of the DNA was determined by the thermal denaturation method (Marmur & Doty, 1962) using a 2168 International Journal of Systematic and Evolutionary Microbiology 65

3 Pullulanibacillus pueri sp. nov. Table 1. Differential characteristics of strain YN3 T and its closest phylogenetic relatives Strain: 1, YN3 T ;2,Pullulanibacillus naganoensis DSM T ;3,Pullulanibacillus uraniitolerans DSM T. All data were from this study. +, Positive; 2, negative; W, weakly positive. Characteristic Colony morphology (margins) Entire Entire Undulate Cell length (mm) Temperature for growth (8C) Range Optimum ph Range Optimum NaCl tolerance (%, w/v) Motility DNA G+C content (mol%) Major cellular fatty acids anteiso-c 15:0, anteiso-c 17:0, anteiso-c 15:0, anteiso-c 17:0, anteiso-c 15:0, anteiso-c 17:0 C 18:1 v7c iso-c 15:0, iso-c 16:0 Presence of: Alkaline phosphatase Valine arylamidase Cystine arylamidase Trypsin W 2 + a-chymotrypsin N-Acetyl-b-glucosaminidase W 2 + a-galactosidase a-fucosidase Esterase (C4) Hydrolysis of: Pullulan Hippurate Starch Xylan CM-cellulose Gelatin Pectin 2 W + Salicin Utilization of: Xylose Mannose Cellobiose Mannitol Glycerol Ribose Rhamnose Sorbitol Xylitol L-Arabitol Fructose Trehalose Lactate Fumarate L-Ornithine L-Serine L-Histidine D-Asparagine L-Valine

4 L. Niu and others 0.01 Sporolactobacillus nakayamae subsp. nakayamae JCM 3514 T (AJ634663) Sporolactobacillus nakayamae subsp. racemicus JCM 3417 T (AJ698860) Sporolactobacillus laevolacticus ATCC T (D16270) 93 Sporolactobacillus kofuensis JCM 3419 T (AB374517) Sporolactobacillus inulinus DSM T (AB101595) Sporolactobacillus terrae ATCC T (AJ634662) Sporolactobacillus putidus JCM T (AB374522) Sporolactobacillus vineae JCM T (EF581819) Pullulanibacillus pueri JCM T (KF733796) Pullulanibacillus naganoensis ATCC T (AB021193) Pullulanibacillus uraniitolerans DSM T (AM931441) Tuberibacillus calidus DSM T (AB231786) Bacillus subtilis subsp. subtilis ATCC 6051 T (AJ276351) Sinobaca qinghaiensis DSM T (DQ168584) Lactobacillus delbrueckii ATCC 9649 T (AY773949) Fig. 1. Phylogenetic dendrogram of strain YN3 T and type strains of related species based on 16S rrna gene sequences. The tree rooted with Lactobacillus delbrueckii ATCC 9649 T was reconstructed using the neighbour-joining method. Bootstrap values (percentages of 0 replications).70 % are shown at nodes. Bar, 0.01 nt substitutions per site. model Cary Bio UV/VIS-spectrophotometer equipped with a Peltier-thermostatted 666 multi-cell changer and a temperature controller with in situ temperature probe (Varian), with Escherichia coli K-12 as the reference. The G+C content of the genomic DNA of strain YN3 T was 38.7 mol%, which was very similar to the values reported for recognized members of the genus Pullulanibacillus (39 mol%) (Pereira et al., 2013). For chemotaxonomic analyses, the biomass of the newly isolated strain and the reference type strains were harvested from CYC agar plates incubated overnight at 30 8C. The diagnostic isomers of diaminopimelic acid and amino acids in the cell wall were determined with established TLC procedures (Lechevalier & Lechevalier, 1980). Cellular fatty acids were extracted, methylated and analysed using the standard MIDI (Microbial Identification) system (Miller, 1982; Sasser, 1990). Isoprenoid quinones were extracted with chloroform/methanol (2:1, v/v), purified by using TLC, and the identities of quinones were analysed by HPLC with an eclipse XDB-C18 column ( mm; Agilent) (Collins, 1985). Polar lipids were extracted and separated by two-dimensional TLC and identified by spraying the TLC plates with appropriate detection reagents (Komagata & Suzuki, 1987, Minnikin et al., 1984). The major cellular fatty acids of strain YN3 T included anteiso-c 17:0 (42.1 %), anteiso-c 15:0 (14.6 %) and C 18:1 v7c (17.1 %). The unsaturated fatty acid C 18:1 v7c was detected as a major component in strain YN3 T, but as a minor component in P. naganoensis DSM T and P. uraniitolerans DSM T (Table S2). The predominant isoprenoid quinone of strain YN3 T was determined to be MK-7, which corresponded to the characteristic quinone found in members of the genus Pullulanibacillus. Strain YN3 T had a cell-wall type A1c peptidoglycan with meso-diaminopimelic acid as the diagnostic diamino acid plus alanine and glutamic acid, but did not contain glucose, galactose and rhamnose, which supported the affiliation of strain YN3 T to the genus Pullulanibacillus. The polar lipid profile of strain YN3 T consisted of phosphatidylglycerol, two unidentified glycolipids, three unidentified aminolipids and two unidentified phospholipids (Fig. S3). In conclusion, phylogenetic and chemotaxonomic characteristics suggested that strain YN3 T belonged to the genus Pullulanibacillus. Furthermore, the DNA DNA relatedness between strain YN3 T and the type strains of recognized species of the genus Pullulanibacillus was low. Physiological and biochemical traits distinguished strain YN3 T from other species of the genus Pullulanibacillus (Table 1). Based on the results presented, strain YN3 T represents a novel species of the genus Pullulanibacillus, for which the name Pullulanibacillus pueri sp. nov. is proposed. Description of Pullulanibacillus pueri sp. nov. Pullulanibacillus pueri (pu9e.ri. N.L. n. puerum Pu er, a tea from China; N.L. gen. n. pueri of Pu er) International Journal of Systematic and Evolutionary Microbiology 65

5 Pullulanibacillus pueri sp. nov. Cells are Gram-stain-positive, straight or slightly curved rods with somewhat pointed ends, mm in width and mm in length. Cells occur singly or in clusters and exhibit twitching motility due to being peritrichously flagellated. Occasionally, sub-terminal elliptical spores are formed. Colonies on CYC agar are yellow, circular, and slightly convex with a diameter of 3.0 mm after cultivation at 30 uc for 48 h. Growth occurs between 20 uc and 50 uc (optimum uc) and at ph (optimum ph ). Growth occurs at NaCl concentrations in the range 0 9 % (w/v). Nitrate is not reduced to nitrite. Positive result in tests for the presence of alkaline phosphatase, esterase(c4), trypsin, a-galactosidase, a-chymotrypsin, a- mannosidase, N-acetyl-b-glucosaminidase, a-fucosidase, valine arylamidase and cystine arylamidase (API ZYM system). Positive result in tests for the hydrolysis of casein, gelatin, salicin and amygdalin, but negative result for the hydrolysis of pullulan, xylan, CM-cellulose, starch, hippurate and pectin. Positive reaction in tests for the assimilation of xylose, mannose, rhamnose, trehalose, fructose, ribose, glycogen, mannitol, sorbitol, inositol, L-arabitol, lactate, L-serine and L-valine, but negative reaction for the assimilation of glycerol, xylitol, methanol, fumarate, citrate, cellobiose, L-ornithine, L-cysteine, L-threonine, L- leucine, L-glutamine, L-tyrosine, L-isoleucine, L-arginine, D-asparagine and L-histidine. The predominant fatty acids are anteiso-c 17:0, anteiso-c 15:0 and C 18:1 v7c. The polar lipid profile consists of phosphatidylglycerol, two unidentified glycolipids, three unidentified aminolipids and two unidentified phospholipids. The major respiratory lipoquinone is MK-7. Cell-wall type is A1c peptidoglycan with meso-diaminopimelic acid. The type strain YN3 T (5CGMCC T 5JCM T ) was isolated from a ripened Pu er tea sample obtained from a manufacturer in Puer City, Yunnan province, China. The DNA G+C content of the type strain is 38.7 mol%. Acknowledgements This study was supported, in part, by the National Natural Science Foundation of China (grant no ), Anhui Provincial Natural Science Foundation (grant no MC31), a grant from the Natural Science Foundation of Jiangsu Province, China (code BK ), as well as a project funded by the Technology Innovation Fund for Small and Medium-sized Enterprises of Jiangsu Province, China (code BC ). We would like to thank Peter Schumann for the analysis of peptidoglycan (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany). References Collins, M. D. (1985). Isoprenoid quinone analysis in classification and identification. In Chemical Methods in Bacterial Systematics, pp Edited by M. Goodfellow & D. E. Minnikin. London: Academic Press. 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