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Kushneria Sinocarnis Sp. Nov., a Moderately Halophilic Bacterium Isolated from a Chinese Traditional Cured-Meat

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Kushneria Sinocarnis Sp. Nov., a Moderately Halophilic Bacterium Isolated from a Chinese Traditional Cured-Meat %paper no. ije013797 charlesworth ref: ije013797& New Taxa - Proteobacteria International Journal of Systematic and Evolutionary Microbiology (2010), 60, 000–000 DOI 10.1099/ijs.0.013797-0 Kushneria sinocarnis sp. nov., a moderately halophilic bacterium isolated from a Chinese traditional cured-meat Zhengzhong Zou and Gejiao Wang Correspondence State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Gejiao Wang Huazhong Agricultural University, Wuhan 430070, PR China [email protected] or [email protected] A Gram-negative, aerobic, moderately halophilic bacterium, designated strain Z35T, was isolated from a Chinese traditional cured-meat produced in Wuhan. The isolate grew with 1–20 % NaCl (optimum 10 %), at 4–42 6C (optimum 37 6C) and at pH 4.5–8.5 (optimum pH 7.0). Cells of strain Z35T were not motile and were rod or oval shaped. The genomic DNA G+C content was 59.1 mol%. The isoprenoid quinones were Q-9 (88.96 %), Q-8 (9.46 %) and Q-10 (1.58 %). The major fatty acids were C19 : 0 cyclo v8c,C16 : 0 and C17 : 0 cyclo. Phylogenetic analysis based on 16S rRNA gene sequence analysis indicated that strain Z35T was closely related to the type strains of Kushneria species with 93.7–95.3 % sequence similarities. Phylogenetic analysis based on 23S rRNA gene sequence similarity values also confirmed the phylogenetic position of the isolate. Therefore, based on the phenotypic, chemotaxonomic and phylogenetic evidences, strain Z35T is affiliated to Kushneria, but is clearly differentiated from other species of this genus and represents a novel member, for which the name Kushneria sinocarnis sp. nov. is proposed. The type strain is Z35T (5CCTCC AB 209027T 5NRRL B-59197T 5DSM 23229T). The family Halomonadaceae, within the Gammaproteo- Recently, Sa´nchez-Porro et al. (2009) described a new genus, bacteria, was proposed by Franzmann et al. (1988). Because Kushneria, with the type species Kushneria aurantia,and of the diversity of the Halomonadaceae and the lack of reclassified Halomonas marisflavi (Yoon et al., 2001), typical differential phenotypic traits, many Gram-negative Halomonas indalinina (Cabrera et al., 2007) and halophilic micro-organisms that were previously classified Halomonas avicenniae (Soto-Ramirez et al., 2007) as as members of genera Deleya, Arthrobacter, Flavobacterium, Kushneria marisflavi, Kushneria indalinina and Kushneria Volcaniella, Halovibrio, Paracoccus, Alcaligenes and Pseudo- avicenniae, respectively. The distinct characteristics of the monas are now assigned to the family Halomonadaceae genus were being oxidase-negative and having small (Ventosa et al., 1998). Based on 16S and 23S rRNA gene amounts of Q-8 and Q-10 in the respiratory quinone sequence relationships and phenotypic features, this system relative to the most closely related genus, Halomonas. heterogeneous group was re-evaluated (Arahal et al., The majority of members of the family Halomonadaceae 2002; Dobson et al., 1993; Mata et al., 2002). At the time were isolated from marine and saline environments of writing, there were nine genera in the family (Ventosa et al., 1998) and rarely from cured-meat Halomonadaceae: Chromohalobacter, Carnimonas, Cobetia, products. To our knowledge, there are only three species Halomonas, Halotalea, Modicisalibacter, Salinicola, Zymo- within the family Halomonadaceae isolated from this type bacter and Kushneria. Halomonas was the largest genus in of environment: Carnimonas nigrificans (Garriga et al., this family and contained 56 species with validly published 1998) from raw cured-meat products; Halomonas halode- names. Chromohalobacter contains nine species. The six nitrificans (Dobson & Franzmann, 1996) from meat-curing genera, Carnimonas, Cobetia, Halotalea, Modicisalibacter, brines; and Halomonas alimentaria (Yoon et al., 2002) Salinicola and Zymobacter, each contained only one species from a traditional Korean fermented seafood. This study (Arahal & Ventosa, 2008). describes a Gram-negative, moderately halophilic bac- terium, strain Z35T, isolated from a piece of Chinese traditional salty pork that was cured with salt in Wuhan, and concludes that strain Z35T represents a novel member The GenBank/EMBL/DDBJ accession numbers for the 16S and 23S of the genus Kushneria. rRNA gene sequences of strain Z35T are FJ667549 and FJ797433, respectively. For the isolation of strain Z35T, the cured meat was cut A supplementary figure showing a scanning electron micrograph of into small pieces, added to sterile HGM liquid medium T [ 21 strain Z35 is available with the online version of this paper. containing (all l ): 19.5 g MgCl2 .6H2O, 29.0 g 013797 G 2010 IUMS Printed in Great Britain 1 %paper no. ije013797 charlesworth ref: ije013797& Z. Zou and G. Wang MgSO4 .7H2O, 1.1 g CaCl2 .2H2O, 6.0 g KCl, 0.5 g NaBr, system (bioMe´rieux), according to the manufacturer’s 10.0 g (NH4)2SO4, 174.0 g NaCl, 3.0 g yeast extract, with instructions, except that the culture was suspended in 10 mM Tris–HCl buffer to maintain the pH at 7.0; Fairley artificial seawater solution (w/v: 10 % NaCl, 0.594 % et al., 2002] and incubated at 37 uC for 24 h with rotation MgSO4 .7H2O, 0.453 % MgCl2 .6H2O, 0.064 % KCl, at 200 r.p.m. After this enrichment cultivation, an aliquot 0.013 % CaCl2). All phenotypic characterizations were of the solution was diluted with 10 % NaCl solution and done using K. avicenniae MW2aT as a reference (Soto- streaked onto HGM agar plates and incubated at 37 uC for Ramirez et al., 2007). 2 weeks. Single colonies were subcultivated several times to The nearly full-length 16S rRNA gene sequence was obtain a pure culture. amplified by PCR with universal primers Uni-27F and For taxonomic characterization, strain Z35T was grown in Uni-1492R (Wilson et al., 1990) and the PCR products MH medium (Ventosa et al., 1982) at 37 uC unless were ligated into the pGEM-T vector (Promega). The full- otherwise stated. Cell morphology was observed using a length 23S rRNA gene sequence was obtained by PCR with scanning electron microscope (JSM-6390/LV; JEOL) with two sets of primer pairs: 1310V (59-GAGTCTGCAA- 20 kV accelerating voltage and at magnification 610 000. CTCGACT-39, forward, positions 1310–1326, Escherichia Before observation, bacterial colonies from the plates were coli 16S rRNA gene sequence numbering; this work) and < bound to slides and fixed with 2.5 % (v/v) glutaraldehyde 985R (59-CCGGTCCTCTCGTACT-39, reverse; Arahal solution. After washing, cells were dehydrated with ethanol et al., 2002); and 1023V (59-GCGTAAYAGCTCACT-39, and coated with gold. forward) and 504R (59-SWGTTCGRVAWGGGA-39, ; reverse) (Arahal et al., 2002). DNA sequencing was The optimal conditions for growth were determined in MH performed at the Beijing Genomics Institute (Beijing, PR medium containing 0, 0.5, 1, 3, 5, 7.5, 10, 12.5, 15, 20, 25 China). The sequences were initially compared with the and 30 % (w/v) total salts, as well as in MH medium in sequences in the GenBank database. Subsequently, the 16S which only the NaCl concentration was changed. The and 23S rRNA gene sequences were aligned with those of specific ionic requirement was examined in MH medium 2+ 2+ + closely related strains of the family Halomonadaceae using without Mg ,Ca and/or K . Growth was also tested the CLUSTAL W algorithm with default parameters with NaCl substituted by KCl. The pH range for growth (Thompson et al., 1994). Phylogenetic analysis was carried was determined in filter-sterilized MH medium buffered out using MEGA 4.0 (Tamura et al., 2007). Distances and with hydrogen acetate/sodium acetate (pH 4.0–4.5), clustering were determined using the neighbour-joining 10 mM MES (pH 5.0–6.0), 10 mM PIPES (pH 6.5–7.0), and maximum-parsimony methods with bootstrap ana- 10 mM Tris/HCl (7.5–9.0) and adjusted with NaOH or lyses based on 1000 replications. HCl. The pH range for growth was tested at pH 4.0–10.0 in The genomic DNA G+C determination and chemotaxo- increments of pH 0.5. Growth was determined at A600. T Growth at 0, 4, 15, 20, 28, 37, 42 and 45 uC was also nomic study of strain Z35 , including whole-cell fatty acid determined. and isoprenoid quinone analyses, were carried out by the China Center for Type Culture Collection (Wuhan, PR Phenotypic characterizations were performed following the China). G+C content was determined by HPLC according recommended minimal standards for describing new taxa to the method of Mesbah et al. (1989). Respiratory of the family Halomonadaceae (Arahal et al., 2007). Gram- lipoquinone analysis was performed by HPLC as described staining reaction was determined using the method by Xie & Yokota (2003). For whole-cell fatty acid analysis, described by Dussault (1955). Growth under anaerobic strain Z35T and the type strains of the genus Kushneria conditions was studied in semi-solid MH medium (0.6 %, were grown on SW10 agar at 37 uC for 48 h supplemented = w/v, agar) by inoculation at the bottom of the tube, sealing with 10 % NaCl and analysed by GC (Hewlett Packard with 2 ml agar (2 %, w/v) and 2 ml paraffin. Acid 6890) according to the Sherlock Microbial Identification production from carbohydrates was determined using System (MIDI) instructions. Fatty acids in strain Z35T after bromcresol purple supplemented with 1 % of the car- growth on TSB agar were also analysed. bohydrate and liquid MH medium. Nutritional tests were T analysed according to Arahal et al. (2007). Substrates were The cell size and morphology of strain Z35 is shown in added as filter-sterilized solutions to basal medium [(all Supplementary Fig. S1 (available in IJSEM Online). 21 Detailed results of the morphological, physiological and l ): 75 g NaCl, 2 g KCl, 0.2 g MgSO4 .7H2O, 1 g KNO3, T ] biochemical characteristics of strain Z35 are given in the 1 g (NH4)2HPO4, 0.5 g KH2PO4 to give a final concen- T 21 species description.
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