International Journal of Systematic and Evolutionary Microbiology (2013), 63, 3769–3776 DOI 10.1099/ijs.0.051607-0 Taibaiella smilacinae gen. nov., sp. nov., an endophytic member of the family Chitinophagaceae isolated from the stem of Smilacina japonica, and emended description of Flavihumibacter petaseus Lei Zhang,1,2 Yang Wang,3 Linfang Wei,1 Yao Wang,1 Xihui Shen1 and Shiqing Li1,2 Correspondence 1State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Xihui Shen Northwest A&F University, Yangling, Shaanxi 712100, PR China [email protected] 2State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Institute of Soil and Water Conservation, Chinese Academy of Sciences and Northwest A&F University, Yangling, Shaanxi 712100, PR China 3Hubei Institute for Food and Drug Control, Wuhan 430072, PR China A light-yellow-coloured bacterium, designated strain PTJT-5T, was isolated from the stem of Smilacina japonica A. Gray collected from Taibai Mountain in Shaanxi Province, north-west China, and was subjected to a taxonomic study by using a polyphasic approach. The novel isolate grew optimally at 25–28 6C and pH 6.0–7.0. Flexirubin-type pigments were produced. Cells were Gram-reaction-negative, strictly aerobic, rod-shaped and non-motile. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain PTJT-5T was a member of the phylum Bacteroidetes, exhibiting the highest sequence similarity to Lacibacter cauensis NJ-8T (87.7 %). The major cellular fatty acids were iso-C15 : 0, iso-C15 : 1 G, iso-C17 : 0 and iso-C17 : 0 3-OH. The only polyamine was homospermidine and the major polar lipid was phosphatidylethanolamine. The only respiratory quinone was MK-7 and the DNA G+C content was 40.3 mol%. Based on the phenotypic, phylogenetic and genotypic data, strain PTJT-5T is considered to represent a novel species of a new genus in the family Chitinophagaceae, for which the name Taibaiella smilacinae gen. nov., sp. nov. is proposed. The type strain of Taibaiella smilacinae is PTJT-5T (5CCTCC AB 2013017T5KCTC 32316T). An emended description of Flavihumibacter petaseus is also proposed. The family Chitinophagaceae within the phylum Bacteroidetes unexplored as potential sources of novel species and novel was proposed by Ludwig et al. (2008) and its description was natural products (Strobel & Daisy, 2003). During the formally established by Ka¨mpfer et al. (2011). At the time of course of a study of the diversity of culturable bacterial writing, the family Chitinophagaceae includes 14 genera: communities associated with traditional Chinese medicinal Chitinophaga (Ka¨mpfer et al., 2006), Ferruginibacter (Lim plants, strain PTJT-5T was isolated from the stem of et al.,2009),Filimonas (Shiratori et al.,2009),Flavihumi- Smilacina japonica A. Gray. Comparative analysis of 16S bacter (Zhang et al., 2010), Flavisolibacter (Yoon & Im, rRNA gene sequences showed that strain PTJT-5T had 2007), Flavitalea (Wang et al., 2011), Hydrotalea (Ka¨mpfer ,88 % sequence similarity to all recognized bacterial et al., 2011), Lacibacter (Qu et al., 2009), Niabella (Kim et al., species, with Lacibacter cauensis NJ-8T as its closest 2007), Niastella (Weon et al., 2006), Parasegetibacter (Zhang phylogenetic relative (87.7 % sequence similarity). Data et al., 2009), Sediminibacterium (Qu & Yuan, 2008), Segeti- from further polyphasic taxonomic study indicate that this bacter (An et al., 2007) and Terrimonas (Xie & Yokota, 2006). strain represents a novel species of a new genus within the family Chitinophagaceae. Endophytes residing within plant tissues are considered to be a rich source of genetic diversity, and remain relatively An apparently healthy plant sample of Smilacina japonica A. Gray was collected from Taibai Mountain The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene (33u 579 N 107u 459 E) in Shaanxi Province, north-west T sequence of strain PTJT-5 is KC571459. China, stored in the dark at 4 uC and subjected to isolation A supplementary figure is available with the online version of this paper. of endophytic bacteria within 48 h. The plant was washed Downloaded from www.microbiologyresearch.org by 051607 G 2013 IUMS Printed in Great Britain 3769 IP: 113.200.245.47 On: Mon, 21 Dec 2015 07:34:06 L. Zhang and others carefully in running water to remove external soil and the maximum-parsimony trees were essentially the same as roots, stems and leaves were separated. After drying at that of the neighbour-joining tree (not shown). These room temperature, the tissue segments were subjected to results suggest that strain PTJT-5T represents a novel genus surface sterilization by using the following steps: a 1 min within the family Chitinophagaceae. wash in 70 % ethanol followed by a 3 min wash in sodium 2 Cell morphology was examined by transmission electron hypochlorite solution (2 % available Cl ), a 0.5 min wash microscopy (Hitachi HT7700) with cells grown for 36 h at in 70 % ethanol and two rinses in sterilized distilled water. 28 uC on 0.16 TSA. Cells were negatively stained with 1 % After surface disinfection, the leaf, stem or root tissue was (w/v) phosphotungstic acid and grids were examined after cut into small fragments and macerated using a sterile pestle and mortar in sterile distilled water. The macerated being air-dried. Gram staining of cells was carried out samples were serially diluted in sterile distilled water, according to the classical Gram procedure described spread on 0.16 trypticase soy agar (TSA; Difco) or R2A by Doetsch (1981). Gliding motility was determined as agar (Difco) supplemented with 50 mg Imazalil ml21 and described by Bowman (2000). The presence of flexirubin- incubated at 25 uC for 2 weeks. Isolate PTJT-5T was type pigments was detected using the bathochromatic shift obtained from a stem sample inoculated on 0.16 TSA, test with 20 % (w/v) KOH (McCammon & Bowman, 2000; routinely cultivated on the same medium at 28 uC and Bernardet et al., 2002). Growth at 4, 7, 10, 15, 25, 28, 30, maintained as a glycerol suspension (20 %, v/v, in distilled 33, 37 and 42 uC was investigated on 0.16 TSA for up to water) at 280 uC. 1 week. Tolerance of NaCl was determined on 0.16 TSA supplemented with 0–5.0 % (w/v) NaCl at 1.0 % intervals. Extraction of genomic DNA was performed with a The pH range and optimum for growth were tested in commercial genomic DNA extraction kit (ChaoShi-Bio). 0.16 trypticase soy broth (TSB) adjusted to pH 4.0–11.0 The 16S rRNA gene was amplified by PCR using the primer at intervals of 1 pH unit (Xu et al., 2005). Growth under pair 27f and 1492r (Lane, 1991) and sequencing of the anaerobic conditions was determined after 1 week of purified PCR product was carried out as described by Lin incubation on 0.16 TSA in an anaerobic jar by using et al. (2004). Identification of phylogenetic neighbours and AnaeroGen anaerobic system envelopes (Oxoid). In calculation of pairwise 16S rRNA gene sequence similarities addition to 0.16 TSA and R2A, growth was also evaluated were achieved using the EzTaxon-e server (http://eztaxon- at 28 uC on TSA, nutrient agar (NA), marine agar 2216 e.ezbiocloud.net/; Kim et al., 2012b). Multiple sequence (MA) and MacConkey agar (all from Difco). Oxidase alignments were performed with CLUSTAL_X (Thompson activity was determined using 1 % (w/v) tetramethyl p- et al., 1997). Phylogenetic trees were reconstructed by using phenylenediamine and catalase activity was determined by the neighbour-joining (Saitou & Nei, 1987) and maximum- assessing bubble production in 3 % (v/v) H2O2. Hydrolysis parsimony (Fitch, 1971) methods in MEGA version 4.1 of starch (0.2 %, w/v; Bodi Chemical Co.), chitin from crab software (Tamura et al., 2007) with bootstrap values based shells (1 %, w/v; Sigma), casein (5 % skimmed milk, w/v; on 1000 replications (Felsenstein, 1985). The evolutionary Difco), CM-cellulose (0.5 %, w/v; Sinopharm Chemical distance matrix for the neighbour-joining method was Reagent Co.) and L-tyrosine (0.5 %, w/v; BBI) was tested by generated according to Kimura’s two-parameter model using 0.16 TSA as the basal medium according to the (Kimura, 1980) and the close-neighbour-interchange algo- methods of Kim et al. (2012a). Hydrolysis of gelatin and rithm (search level52, random additions5100) was used in aesculin was determined according to the methods of the maximum-parsimony method. Relationships among Smibert & Krieg (1994). Activities of constitutive enzymes taxa were also established with the maximum-likelihood and other physiological properties were determined by algorithm in the PHYLIP package version 3.66 (Felsenstein, using the API 20E, API 20NE and API ZYM strips 2006). (bioMe´rieux) according to the manufacturer’s instructions, The nearly complete 16S rRNA gene sequence (1397 bp) of except that the incubation temperature was 28 uC. T strain PTJT-5 was determined and subjected to phylo- T T Cells of strain PTJT-5 were Gram-reaction-negative, genetic analysis, which revealed that strain PTJT-5 was strictly aerobic, non-motile and rod-shaped. Other pheno- most closely related to members of the family Chitino- typic features of strain PTJT-5T are summarized in the phagaceae. The closest relative of strain PTJT-5T was T genus and species descriptions. Differential characteristics Lacibacter cauensis NJ-8 , but the sequence similarity of the novel strain and type species of genera of the family between them was only 87.7 %. Lower sequence similarities Chitinophagaceae are shown in Table 1. Strain PTJT-5T (83.3–87.3 %) were found to the type strains of described could be distinguished clearly from its closest phylogenetic species of the genera Chitinophaga, Ferruginibacter, Nia- T relative Lacibacter cauensis NJ-8 by a number of pheno- bella, Segetibacter, Terrimonas, Sediminibacterium, Flavi- typic characteristics, including production of flexirubin- talea, Niastella, Filimonas, Flavisolibacter, Parasegeti- type pigments, temperature range for growth, nitrate bacter, Flavihumibacter and Hydrotalea.